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Introduction

Seventeen years have passed since the outbreak of severe acute respiratory syndrome (SARS) in 2003, but there is yet no approved treatment for infections with the SARS coronavirus (SARS-CoV). (1) One of the reasons is that, despite the devastating consequences of SARS for the affected patients, the development of an antiviral drug against this virus would not be commercially viable in view of the fact that the virus has been rapidly contained and did not reappear since 2004. As a result, we were empty-handed when the Middle East respiratory syndrome coronavirus (MERS-CoV), a close relative of SARS-CoV, emerged in 2012. (2) MERS is characterized by severe respiratory disease, quite similar to SARS, but in addition, frequently causes renal failure. (3) Although the number of registered MERS cases is low (2494 as of November 30, 2019; www.who.int), the threat MERS-CoV poses to global public health may be even more serious than that presented by SARS-CoV. This is related to the high case-fatality rate (about 35%, compared to 10% for SARS) and to the fact that MERS cases are still accumulating seven years after the discovery of the virus, whereas the SARS outbreak was essentially contained within 6 months. The potential for human-to-human transmission of MERS-CoV has been impressively demonstrated by the 2015 outbreak in South Korea, where 186 cases could be traced back to a single infected traveler returning from the Middle East. (4) SARS-like coronaviruses are still circulating in bats in China, (5−8) from where they may spill over into the human population; this is probably what caused the current outbreak of atypical pneumonia in Wuhan, which is linked to a seafood and animal market. The RNA genome (GenBank accession code: MN908947.3; http://virological.org/t/initial-genome-release-of-novel-coronavirus/319, last accessed on January 11, 2020) of the new betacoronavirus features around 82% identity to that of SARS-CoV.

In spite of the considerable threat posed by SARS-CoV and related viruses, as well as by MERS-CoV, it is obvious that the number of cases so far does not warrant the commercial development of an antiviral drug targeting MERS- and SARS-CoV even if a projected steady growth of the number of MERS cases is taken into account. A possible solution to the problem could be the development of broad-spectrum antiviral drugs that are directed against the major viral protease, a target that is shared by all coronavirus genera as well as, in a related form, by members of the large genus Enterovirus in the picornavirus family. Among the members of the genus Alphacoronavirus are the human coronaviruses (HCoV) NL63 (ref (9)) and 229E (10) that usually cause only mild respiratory symptoms in otherwise healthy individuals, but are much more widespread than SARS-CoV or MERS-CoV. Therapeutic intervention against alphacoronaviruses is indicated in cases of accompanying diseases such as cystic fibrosis (11) or leukemia, (12) or certain other underlying medical conditions. (13) The enteroviruses include pathogens such as EV-D68, the causative agent of the 2014 outbreak of the "summer flu" in the U.S., (14) EV-A71 and Coxsackievirus A16 (CVA16), the etiological agents of hand, foot, and mouth disease (HFMD), (15) Coxsackievirus B3 (CVB3), which can cause myocardic inflammation, (16) and human rhinoviruses (HRV), notoriously known to lead to the common cold but also capable of causing exacerbations of asthma and COPD. (17) Infection with some of these viruses can lead to serious outcomes; thus, EV-D68 can cause polio-like disease, (18) and EV-A71 infection can proceed to aseptic meningitis, encephalitis, pulmonary edema, viral myocarditis, and acute flaccid paralysis. (15,19,20) Enteroviruses cause clinical disease much more frequently than coronaviruses so that an antiviral drug targeting both virus families should be commercially viable.

However, enteroviruses are very different from coronaviruses. While both of them have a single-stranded RNA genome of positive polarity, that of enteroviruses is very small (just 7–9 kb), whereas coronaviruses feature the largest RNA genome known to date (27–34 kb). Enteroviruses are small, naked particles, whereas coronaviruses are much larger and enveloped. Nevertheless, a related feature shared by these two groups of viruses is their type of major protease, (21) which in the enteroviruses is encoded by the 3C region of the genome (hence the protease is designated 3C^pro). In coronaviruses, nonstructural protein 5 (Nsp5) is the main protease (M^pro). Similar to the enteroviral 3C^pro, it is a cysteine protease in the vast majority of cases and has, therefore, also been called a ″3C-like protease" (3CL^pro). The first crystal structure of a CoV M^pro or 3CL^pro (ref (22)) revealed that two of the three domains of the enzyme together resemble the chymotrypsin-like fold of the enteroviral 3C^pro, but there is an additional α-helical domain that is involved in the dimerization of the protease (Figure 1A). This dimerization is essential for the catalytic activity of the CoV M^pro, whereas the enteroviral 3C^pro (Figure 1B) functions as a monomer. Further, the enteroviral 3C^pro features a classical Cys···His···Glu/Asp catalytic triad, whereas the CoV M^pro only has a Cys···His dyad. (22) Yet, there are a number of common features shared between the two types of proteases, in particular their almost absolute requirement for Gln in the P1 position of the substrate and space for only small amino-acid residues such as Gly, Ala, or Ser in the P1′ position, encouraging us to explore the coronaviral M^pro and the enteroviral 3C^pro as a common target for the design of broad-spectrum antiviral compounds. The fact that there is no known human protease with a specificity for Gln at the cleavage site of the substrate increases the attractiveness of this viral target, as there is hope that the inhibitors to be developed will not show toxicity versus the host cell. Indeed, neither the enterovirus 3C^pro inhibitor rupintrivir, which was developed as a treatment of the common cold caused by HRV, nor the peptide aldehyde inhibitor of the coronavirus M^pro that was recently demonstrated to lead to complete recovery of cats from the normally fatal infection with Feline Infectious Peritonitis Virus (FIPV) showed any toxic effects on humans or cats, respectively. (23,24)

Figure 1. Crystal structures of the SARS-CoV main protease (M^pro, ref (26); PDB entry 2BX4) and Coxsackivirus B3 3C protease (3C^pro; Tan et al., unpublished; PDB entry 3ZYD). Catalytic residues are indicated by spheres (yellow, Cys; blue, His; red, Glu). (A) The coronavirus M^pro is a homodimer, with each monomer comprising three domains. (B) The structure of the monomeric CVB3 3C^pro resembles the N-terminal two domains of the SARS-CoV M^pro. The structure is on the same scale as image A. (C) Superimposition of residues from the two structures involved in ligand binding. Superimposition was carried out by aligning the catalytic Cys-His pair of each protease. Residues of the SARS-CoV M^pro are shown with carbon atoms in cyan, and CVB3 3C^pro residues have orange carbons and are labeled with an asterisk (*).

We chose the chemical class of peptidomimetic α-ketoamides to assess the feasibility of achieving antiviral drugs targeting coronaviruses and enteroviruses with near-equipotency. Here we describe the structure-based design, synthesis, and evaluation of the inhibitory activity of a series of compounds with broad-spectrum activities afforded by studying the structure–activity relationships mainly with respect to the P2 position of the peptidomimetics. One of the compounds designed and synthesized exhibits excellent activity against MERS-CoV.

Results

Structure-Based Design of α-Ketoamides

Our efforts to design novel α-ketoamides as broad-spectrum inhibitors of coronavirus M^pros and enterovirus 3C^pros started with a detailed analysis of the following crystal structures of unliganded target enzymes: SARS-CoV M^pro (refs (25−27); PDB entries 1UJ1, 2BX3, 2BX4); bat coronavirus HKU4 M^pro as a surrogate for the closely related MERS-CoV protease (our unpublished work (Ma, Xiao et al.; PDB entry 2YNA; see also ref (27)); HCoV-229E M^pro (refs (27and28); PDB entry 1P9S); Coxsackievirus B3 3C^pro (our unpublished work; Tan et al., PDB entry 3ZYD); enterovirus D68 3C^pro (ref (29); PDB entry 3ZV8); and enterovirus A71 3C^pro (ref (30); PDB entry 3SJK). During the course of the present study, we determined crystal structures of a number of lead α-ketoamide compounds in complex with SARS-CoV M^pro, HCoV-NL63 M^pro, and CVB3 3C^pro, in support of the design of improvements in the next round of lead optimization. Notably, unexpected differences between alpha- and betacoronavirus M^pro were found in this study. The structural foundation of these was elucidated in detail in a subproject involving the M^pro of HCoV NL63; because of its volume, this work will be published separately (Zhang et al., in preparation) and only some selected findings are referred to here. The main protease of the newly discovered coronavirus linked to the Wuhan outbreak of respiratory illness is 96% identical (98% similar) in the amino-acid sequence to that of SARS-CoV M^pro (derived from the RNA genome of BetaCoV/Wuhan/IVDC-HB-01/2019, Genbank accession code: MN908947.3; http://virological.org/t/initial-genome-release-of-novel-coronavirus/319, last accessed on January 11, 2020), so all results reported here for the inhibition of SARS-CoV will most likely also apply to the new virus.

As the proteases targeted in our study all specifically cleave the peptide bond following a P1-glutamine residue (HCoV-NL63 M^pro uniquely also accepts P1 = His at the Nsp13/Nsp14 cleavage site (31)), we decided to use a 5-membered ring (γ-lactam) derivative of glutamine (henceforth called Gln Lactam) as the P1 residue in all our α-ketoamides (see Scheme 1). This moiety has been found to be a good mimic of glutamine and enhance the power of the inhibitors by up to 10-fold, most probably because, compared to the flexible glutamine side chain, the more rigid lactam leads to a reduction of the loss of entropy upon binding to the target protease. (29,32) Our synthetic efforts were, therefore, aimed at optimizing the substituents at the P1′, P2, and P3 positions of the α-ketoamides.

Synthesis of α-Ketoamides

Synthesis (Scheme 1) started with the dianionic alkylation of N-Boc glutamic acid dimethyl ester with bromoacetonitrile. As expected, this alkylation occurred in a highly stereoselective manner, giving 1 as the exclusive product. In the following step, the cyano group of 1 was subjected to hydrogenation. The in situ cyclization of the resulting intermediate afforded the lactam 2. The lactam derivative 3 was generated by removal of the protecting group of 2. On the other hand, the amidation of acyl chloride and α-amino acid methyl ester afforded the intermediates 4, which gave rise to the acids 5 via alkaline hydrolysis. The key intermediates 6 were obtained via the condensation of the lactam derivative 3 and the N-capped amino acids 5. The ester group of compounds 6 was then reduced to the corresponding alcohol. Oxidation of the alcohol products 7 by Dess–Martin periodinane generated the aldehydes 8, followed by nucleophilic addition with isocyanides, gave rise to compounds 9 under acidic conditions. Then, the α-hydroxyamides 10 were prepared by removing the acetyl group of compounds 9. In the final step, the oxidation of the exposed alcohol group in compounds 10 generated our target α-ketoamides 11.

The inhibitory potencies of candidate α-ketoamides were evaluated against purified recombinant SARS-CoV M^pro, HCoV-NL63 M^pro, CVB3 3C^pro, and EV-A71 3C^pro. The most potent compounds were further tested against viral replicons and against SARS-CoV, MERS-CoV, or a whole range of enteroviruses in cell culture-based assays (Tables 1–3 and Supplementary Table 1).

Table 1. Inhibition of Viral Proteases by α-Ketoamides (IC₅₀, μM)

Viral Replicons

To enable the rapid and biosafe screening of antivirals against corona- and enteroviruses, a noninfectious, but replication-competent SARS-CoV replicon was used (33) along with subgenomic replicons of CVB3 (34) and EV-A71 (a kind gift from B. Zhang, Wuhan, China). The easily detectable reporter activity (firefly or Renilla luciferase) of these replicons has previously been shown to reflect viral RNA synthesis. (33−35)In vitro RNA transcripts of the enteroviral replicons were also used for transfection. For the SARS-CoV replicon containing the CMV promoter, only the plasmid DNA was used for transfection.

Initial Inhibitor Design Steps

The initial compound to be designed and synthesized was 11a, which carries a cinnamoyl N-cap in the P3 position, a benzyl group in P2, the glutamine lactam (Gln Lactam) in P1, and benzyl in P1′ (Table 1). This compound showed good to mediocre activities against recombinant SARS-CoV M^pro (IC₅₀ = 1.95 μM; for all compounds, see Tables 1–3 for standard deviations), CVB3 3C^pro (IC₅₀ = 6.6 μM), and EV-A71 3C^pro (IC₅₀ = 1.2 μM), but was surprisingly completely inactive (IC₅₀ > 50 μM) against HCoV-NL63 M^pro. These values were mirrored in the SARS-CoV and in the enterovirus replicons (Table 2). In virus-infected cell cultures, the results obtained were also good to mediocre (Table 3): SARS-CoV (EC₅₀ = 5.8 μM in Vero E6 cells), MERS-CoV (EC₅₀ = 0.0047 μM in Huh7 cells), HCoV 229E (EC₅₀ = 11.8 μM in Huh7 cells), or a host of enteroviruses (EC₅₀ = 9.8 μM against EV-A71 in RD cells; EC₅₀ = 0.48 μM against EV-D68 in HeLa Rh cells; EC₅₀ = 5.6 μM against HRV2 in HeLa Rh cells). In all cell types tested, the compound generally proved to be nontoxic, with selectivity indices (CC₅₀/EC₅₀) usually >10 (Table 3).

Table 2. α-Ketoamide-Induced Inhibition of Subgenomic RNA Synthesis Using Replicons in a Cell-Based Assay (EC₅₀, μM)

Table 3. Cytotoxicity and Antiviral Activity of α-Ketoamides against Selected Entero- and Coronaviruses in a Live-Virus Cell-Based Assay

Crystal structures of compound 11a in complex with SARS-CoV M^pro, HCoV-NL63 M^pro, and CVB3 3C^pro demonstrated that the α-keto-carbon is covalently linked to the active-site Cys (nos. 145, 144, and 147, respectively) of the protease (Figures 2 and 3a–c). The resulting thiohemiketal is in the R configuration in the SARS-CoV and HCoV-NL63 M^pro but in the S configuration in the CVB3 3C^pro complex. The reason for this difference is that the oxygen atom of the thiohemiketal accepts a hydrogen bond from the catalytic His40 in the CVB3 protease, rather than from the main-chain amides of the oxyanion hole as in the SARS-CoV and HCoV-NL63 enzymes (Figure 3a–c insets). It is remarkable that we succeeded in obtaining a crystal structure of compound 11a in complex with the HCoV-NL63 M^pro, even though it has no inhibitory effect on the activity of the enzyme (IC₅₀ > 50 μM) (Figure 2c). Apparently, the compound is able to bind to this M^pro in the absence of the peptide substrate, but cannot compete with the substrate for the binding site due to low affinity. A similar observation has been made in one of our previous studies, where we were able to determine the crystal structure of a complex between the inactive Michael-acceptor compound SG74 and the EV-D68 3C^pro (ref (29); PDB entry 3ZV9).

Figure 2. Fit of compound **11a** (pink carbon atoms) to the target proteases (wheat surfaces) as revealed by X-ray crystallography of the complexes. (A) F_o – F_c difference density (contoured at 3σ) for **11a** in the substrate-binding site of the SARS-CoV M^pro (transparent surface). Selected side chains of the protease are shown with green carbon atoms. (B) Another view of **11a** in the substrate-binding site of the SARS-CoV M^pro. Note the "lid" formed by residue Met49 and its neighbors above the S2 pocket. (C) **11a** in the substrate-binding site of HCoV-NL63 M^pro. Because of the restricted size of the S2 pocket, the P2 benzyl group of the compound cannot enter deeply into this site. Note that the S2 pocket is also covered by a "lid" centered around Thr47. (D) **11a** in the substrate-binding site of the CVB3 3C^pro. The S2 site is large and not covered by a "lid".

Figure 3. Detailed interactions of peptidomimetic α-ketoamides (pink carbon atoms) with target proteases (green carbon atoms). Hydrogen bonds are depicted as blue dashed lines. The inset at the top of the images shows the stereochemistry of the thiohemiketal formed by the nucleophilic attack of the catalytic Cys residue onto the α-keto group. (A) Binding of **11a** to SARS-CoV M^pro. The thiohemiketal is in the R configuration, with its oxygen accepting two hydrogen bonds from the oxyanion-hole amides of Gly143 and Cys145. The amide oxygen accepts an H-bond from His41. The side chains of Ser144 and Arg188 have been omitted for clarity. (B) The P2-benzyl substituent of **11a** cannot fully enter the S2 pocket of the HCoV-NL63 M^pro, which is much smaller and has less plasticity than the corresponding pocket of SARS-CoV M^pro (cf. A). The benzyl, therefore, binds above the pocket in the view shown here; this is probably the reason for the total inactivity (IC₅₀ > 50 μM) of compound **11a** against HCoV-NL63 M^pro. The small size of the pocket is due to the replacement of the flexible Gln189 of the SARS-CoV M^pro by the more rigid Pro189 in this enzyme. The stereochemistry of the thiohemiketal is R. The side chains of Ala143 and Gln188 have been omitted for clarity. (C) Binding of **11a** to the CVB3 3C^pro. The stereochemistry of the thiohemiketal is S, as the group accepts a hydrogen bond from His41, whereas the amide keto group accepts three H-bonds from the oxyanion hole (residues 145–147). The side chain of Gln146 has been omitted for clarity. (D) The crystal structure of **11f** in complex with HCoV-NL63 M^pro shows that this short (inactive) compound lacking a P3 residue has its P2-Boc group inserted into the S2 pocket of the protease. The stereochemistry of the thiohemiketal is S. The side chains of Ala143 and Gln188 have been omitted for clarity. (E) In contrast to P2 = benzyl in **11a**, the isobutyl group of **11n** is small and flexible enough to enter into the narrow S2 pocket of the HCoV-NL63 M^pro. The thiohemiketal is in the R configuration. The side chains of Ala143 and Gln188 have been omitted for clarity. (F) In spite of its small size, the cyclopropylmethyl side chain in the P2 position of **11s** can tightly bind to the S2 subsite of the SARS-CoV M^pro, as this pocket exhibits pronounced plasticity due to the conformational flexibility of Gln189 (see also Figure 4). The stereochemistry of the thiohemiketal is S. The side chains of Ser144 and Arg188 have been omitted for clarity.

P1′ and P3 Substituents

The crystal structures indicated that the fits of the P1′ benzyl group of 11a in the S1′ pocket and of the P3 cinnamoyl cap in the S3 subsite might be improved (see Figure 3a–c). Compounds 11b–11e and 11g–11l were synthesized in an attempt to do so; however, none of them showed better inhibitory activity against the majority of the recombinant proteases, compared to the parent compound, 11a (see Supplementary Results). To investigate whether the P3 residue of the inhibitor is dispensable, we synthesized compound 11f, which only comprises P2 = Boc, P1 = Gln Lactam, and P1′ = benzyl. Compound 11f was inactive against all purified proteases and in all replicons tested but showed some activity against HRV2 in HeLa Rh cells (EC₅₀ = 9.0 μM). A crystal structure of 11f bound to HCoV-NL63 M^pro demonstrated that the P2-Boc group entered the S2 pocket (Figure 3d). In conclusion, although there is probably room for further improvement, we decided to maintain the original design with P1′ = benzyl and P3 = cinnamoyl and focused on improving the P2 substituent.

Properties of the S2 Pockets of the Target Enzymes

The crystal structures of SARS-CoV M^pro, HCoV-NL63 M^pro, and CVB3 3C^pro in complex with 11a revealed a fundamental difference between the S2 pockets of the coronavirus proteases and the enterovirus proteases: The cavities are covered by a "lid" in the former but are open to one side in the latter (Figure 2b–d). In SARS-CoV M^pro, the lid is formed by the 3₁₀ helices 46–51 and in HCoV-NL63 M^pro by the loops 43–48. Residues from the lid, in particular Met49 in the case of SARS-CoV M^pro, can thus make hydrophobic interactions with the P2 substituent of the inhibitor, whereas such interaction is missing in the enterovirus 3C^pros. In addition to the lid, the S2 pocket is lined by the "back wall" (main-chain atoms of residues 186 and 188 and Cβ atom of Asp187), the side walls (Gln189, His41), as well as the "floor" (Met165) in SARS-CoV M^pro. In HCoV-NL63 M^pro, the corresponding structural elements are main-chain atoms of residues 187 and 188 as well as the Cβ atom of Asp187 (back-wall), Pro189 and His41 (side-walls), and Ile165 (floor). Finally, in CVB3 3C^pro, Arg39, Asn69, and Glu71 form the back wall, residues 127–132 and His40 form the side walls, and Val162 constitutes the floor.

In addition, the S2 pocket is of different sizes in the various proteases. The SARS-CoV enzyme features the largest S2 pocket, with a volume of 252 Å³ embraced by the residues (Gln189, His41) defining the side walls of the pocket in the ligand-free enzyme, as calculated by using Chimera, (36) followed by the CVB3 3C^pro S2 pocket with about 180 Å³ (the space between Thr130 and His40). The HCoV-NL63 M^pro has by far the smallest S2 pocket of the three enzymes, with a free space of only 45 Å³ between Pro189 and His41, according to Chimera.

In agreement with these observations, a good fit is observed between the P2 benzyl group of 11a and the S2 subsite of the SARS-CoV M^pro as well as that of the CVB3 3C^pro (Figure 3a,c). In contrast, the crystal structure of the complex between 11a and HCoV-NL63 M^pro, against which the compound is inactive, demonstrates that the P2-benzyl group cannot fully enter the S2 pocket of the enzyme because of the restricted size of this site (Figure 3b).

Thus, the properties of our target proteases with respect to the S2 pocket were defined at this point as "small" and "covered by a lid" for HCoV-NL63 M^pro, "large" and "covered" for SARS-CoV M^pro, and "large" and "open" for CVB3 3C^pro. Through comparison with crystal structures of other proteases of the same virus genus (HCoV-229E M^pro for alphacoronaviruses (28) (PDB entry 1P9S); HKU4-M^pro for betacoronaviruses (Ma, Xiao et al., unpublished; PDB entry 2YNA); and EV-A71 3C^pro for enteroviruses (30) (PDB entry 3SGK), we ensured that our conclusions drawn from the template structures were valid for other family members as well.

To explore the sensitivity of the S2 pocket toward a polar substituent in the para position of the benzyl group, we synthesized compound 11m, carrying a 4-fluorobenzyl group in P2. This substitution abolished almost all activity against the SARS-CoV M^pro (IC₅₀ > 50 μM), and the compound proved inactive against HCoV-NL63 M^pro as well, whereas IC₅₀ values were 2.3 μM against the EV-A71 3C^pro and 8.7 μM against CVB3 3C^pro. From this, we concluded that the introduction of the polar fluorine atom is not compatible with the geometry of the S2 pocket of SARS-CoV M^pro, whereas the fluorine can accept a hydrogen bond from Arg39 in EV-A71 3C^pro (ref (30)) and probably also CVB3 3C^pro. In SARS-CoV M^pro, however, the carbonyl groups of residues 186 and 188 might lead to the repulsion of the fluorinated benzyl group.

P2-Alkyl Substituents of Varying Sizes

As the P2-benzyl group of 11a was apparently too large to fit into the S2 pocket of the HCoV-NL63 M^pro, we replaced it by isobutyl in 11n. This resulted in improved activities against SARS-CoV M^pro (IC₅₀ = 0.33 μM) and in a very good activity against HCoV-NL63 M^pro (IC₅₀ = 1.08 μM, compared with the inactive 11a). For EV-A71 3C^pro, however, the activity decreased to IC₅₀ = 13.8 μM, different from CVB3 3C^pro, where IC₅₀ was 3.8 μM. Our interpretation of this result is that the smaller P2-isobutyl substituent of 11n can still interact with the "lid" (in particular, Met49) of the SARS-CoV M^pro S2 site, but is unable to reach the "back wall" of the EV-A71 3C^pro pocket and thus, in the absence of a "lid", cannot generate sufficient enthalpy of binding. We will see from examples to follow that this trend persists among all inhibitors with a smaller P2 substituent: Even though the SARS-CoV M^pro S2 pocket has a larger volume than that of the enterovirus 3C^pro, the enzyme can be efficiently inhibited by compounds carrying a small P2 residue that makes hydrophobic interactions with the lid (Met49) and floor (Met165) residues.

The EC₅₀ of 11n was >10 μM against the EV-A71 and CVB3 replicons, and even in the SARS-CoV replicon, the activity of 11n was relatively weak (EC₅₀ = 7.0 μM; Table 2). In agreement with the replicon data, 11n proved inactive against EV-A71 in RD cells and showed limited activity against HRV2 or HRV14 in HeLa Rh cells (Table 3). Only the comparatively good activity (EC₅₀ = 4.4 μM) against EV-D68 in HeLa Rh cells was unexpected. The activity of 11n against HCoV 229E in Huh7 cells was good (EC₅₀ = 0.6 μM), and against MERS-CoV in Huh7 cells, it was excellent, with EC₅₀ = 0.0048 μM, while in Vero cells, the EC₅₀ against MERS-CoV was as high as 9.2 μM. Similarly, the EC₅₀ against SARS-CoV in Vero cells was 14.2 μM (Table 3).

We managed to obtain crystals of 11n in complex with the M^pro of HCoV NL63 and found the P2 isobutyl group to be well embedded in the S2 pocket (Figure 3e). This is not only a consequence of the smaller size of the isobutyl group compared to the benzyl group, but also of its larger conformational flexibility, which allows a better fit to the binding site.

When we replaced the P2-isobutyl residue of 11n by n-butyl in 11o, the activities were as follows: IC₅₀ = 8.5 μM for SARS-CoV M^pro, totally inactive (IC₅₀ > 50 μM) against HCoV-NL63 M^pro, IC₅₀ = 3.2 μM for EV-A71 3C^pro, and 5.2 μM for CVB3 3C^pro. The decreased activity in the case of SARS-CoV M^pro and the total inactivity against HCoV-NL63 M^pro indicate that the n-butyl chain is too long for the S2 pocket of these proteases, whereas the slight improvement against EV-A71 3C^pro and CVB3 3C^pro is probably a consequence of the extra space that is available to long and flexible substituents because of the lack of a lid covering the enterovirus 3C^pro pocket.

As the n-butyl substituent in P2 of 11o was obviously too long, we next synthesized a derivative with the shorter propargyl (ethinylmethyl) as the P2 residue (compound 11p). This led to very mediocre activities against all tested proteases. Using cyclopropyl as the P2 residue (compound 11q), the IC₅₀ values were even higher against most of the proteases tested. Obviously, the P2 side chain requires a methylene group in the β-position in order to provide the necessary flexibility for the substituent to be embedded in the S2 pocket.

Modifying Ring Size and Flexibility of P2-Cycloalkylmethyl Substituents

Having realized that, in addition to size, the flexibility of the P2 substituent may be an important factor influencing inhibitory activity, we introduced flexibility into the phenyl ring of 11a by reducing it. The cyclohexylmethyl derivative 11r exhibited an IC₅₀ = 0.7 μM against SARS-CoV M^pro, 12.3 μM against HCoV-NL63 M^pro, 1.7 μM against EV-A71 3C^pro, and 0.9 μM against CVB3 3C^pro. Thus, the replacement of the phenyl group by the cyclohexyl group led to a significant improvement of the inhibitory activity against the recombinant SARS-CoV M^pro and to a dramatic improvement in the case of CVB3 3C^pro. Even for the HCoV-NL63 M^pro, against which 11a was completely inactive, greatly improved, albeit still weak activity was observed (Table 1). In the viral replicons, 11r performed very well, with EC₅₀ = 0.8–0.9 μM for the EV-A71 replicon, 0.45 μM for CVB3, and 1.4 μM for SARS-CoV (Table 2). In the virus-infected cell culture assays (Table 3), 11r exhibited an EC₅₀ = 3.7 μM against EV-A71 in RD cells and an EC₅₀ = 0.48–0.7 μM against EV-D68, HRV2, and HRV14 in HeLa cells. Against HCoV 229E in Huh7 cells, the EC₅₀ was surprisingly low (1.8 μM). Interestingly, the compound proved extremely potent against MERS-CoV in Huh7 cells, with EC₅₀ = 0.0004 μM (400 picomolar). Even in Vero cells, EC₅₀ against MERS-CoV was 5 μM, and the EC₅₀ against SARS-CoV in Vero E6 cells was 1.8–2.1 μM, i.e., the best activity we have seen for an M^pro inhibitor against SARS-CoV in this type of cells. The therapeutic index (CC₅₀/EC₅₀) of 11r against EV-D68, HRV2, and HRV14 was >15 in HeLa Rh cells as well as against CVB3 in Huh-T7 cells, but only ∼5 for EV-A71 in RD cells.

At this point, we decided to systematically vary the size of the ring system in P2. The next substituent to be tried was cyclopropylmethyl (compound 11s, which showed good activities against SARS-CoV M^pro (IC₅₀ = 0.24 μM) and HCoV-NL63 M^pro (1.4 μM), but poor values against EV-A71 3C^pro (IC₅₀ = 18.5 μM) and CVB3 3C^pro (IC₅₀ = 4.3 μM) (Table 1). Compound 11s was shown to inhibit the SARS-CoV replicon with an EC₅₀ of about 2 μM, whereas activity against the EV-A71 and CVB3 replicons was poor (EC₅₀ values > 20 μM) (Table 2). The replicon results were mirrored by the antiviral activity of 11s in enterovirus-infected cells (Table 3), which was weak or very weak. By contrast, the compound inhibited HCoV 229E and MERS-CoV in Huh7 cells with EC₅₀ values of 1.3 and 0.08 μM, respectively. The activity against the latter virus in Vero cells was poor (EC₅₀ ∼ 11 μM), and so was the anti-SARS-CoV activity in Vero E6 cells (Table 3).

We next analyzed the crystal structure of the complex between SARS-CoV M^pro and compound 11s (Figure 3f). The cyclopropylmethyl substituent was found to be incorporated deeply into the S2 pocket, making hydrophobic interactions with Met49 (the lid), Met165 (the floor), and the Cβ of Asp187 (the back wall). In spite of the small size of the P2 substituent, this is possible because the S2 pocket of SARS-CoV M^pro is flexible enough to contract and enclose the P2 moiety tightly. This plasticity is expressed in a conformational change of residue Gln189, both in the main chain and in the side chain. The main-chain conformational change is connected with a flip of the peptide between Gln189 and Thr190. The χ1 torsion angle of the Gln189 side chain changes from roughly antiperiplanar (ap) to (−)-synclinal (sc) (Figure 4). The conformational variability of Gln189 has been noted before, both in molecular dynamics simulations (26) and in other crystal structures. (37) As a consequence of these changes, the side-chain oxygen of Gln189 can accept a 2.54 Å hydrogen bond from the main-chain NH of the P2 residue in the 11s complex (see Figure 4). The affinity of 11s for the S2 pocket of HCoV-NL63 M^pro is good because of an almost ideal match of size and not requiring conformational changes, which this enzyme would not be able to undergo because of the replacement of the flexible Gln189 by the more rigid Pro. On the other hand, docking of the same compound into the crystal structure of the CVB3 3C^pro revealed that the cyclopropylmethyl moiety was probably unable to generate sufficient free energy of binding because of the missing lid and the large size of the S2 pocket in the enterovirus 3C^pro, thereby explaining the poor inhibitory activity of 11s against these targets.

Figure 4. A pronounced plasticity of the S2 pocket of SARS-CoV M^pro is revealed by a comparison of the geometry of the subsite in the complexes with **11a** (P2 = benzyl; inhibitor cyan, protein green) and **11s** (P2 = cyclopropylmethyl; inhibitor orange, protein pink). The main differences here concern the main chain around Gln189 (note the flip of the 189–190 peptide bond) as well as the side chain of this flexible residue, the conformational change of which allows the S2 pocket to "shrink" and adapt to the small size of the P2 substituent in **11s**. This change also enables the formation of a hydrogen bond between the main-chain amide of the P2 residue and the side-chain oxygen of Gln189. The side chains of Arg188 and Thr190, as well as the P1′ substituent of the inhibitors, have been omitted for clarity.

We next introduced cyclobutylmethyl in the P2 position (compound 11t) and obtained the following results: IC₅₀ = 1.4 μM for SARS-CoV M^pro, 3.4 μM for HCoV-NL63 M^pro, 10.8 μM for EV-A71 3C^pro, and 4.8 μM for CVB3 3C^pro (Table 1). Experiments with the viral replicons confirmed this trend, although the EC₅₀ value for SARS-CoV (6.8 μM) was surprisingly high (Table 2). In Huh7 cells infected with MERS-CoV, this compound exhibited EC₅₀ = 0.1 μM (but 9.8 μM in Vero cells), whereas EC₅₀ was 7.0 μM against SARS-CoV in Vero E6 cells. The compound was largely inactive against EV-A71 in RD cells and inhibited the replication of the two HRV subtypes tested (in HeLa Rh cells) with EC₅₀ values of ∼4 μM. The CC₅₀ of 11t in HeLa cells was 65 μM; i.e., the therapeutic index was well above 15 (Table 3).

Obviously, this substituent was still a bit too small for the enterovirus proteases, so as the next step, we tested P2 = cyclopentylmethyl (compound 11u). This turned out to be the one compound with acceptable IC₅₀ values against all tested enzymes: 1.3 μM against SARS-CoV M^pro, 5.4 μM against HCoV-NL63 M^pro, 4.7 μM against EV-A71 3C^pro, and 1.9 μM against CVB3 3C^pro (Table 1). The activity against the replicons was between 3.6 and 4.9 μM (Table 2). In Huh7 cells infected with HCoV 229E or MERS-CoV, 11u showed an EC₅₀ = 2.5 or 0.03 μM (11.1 μM for MERS-CoV in Vero cells), while the EC₅₀ was 4.9 μM against SARS-CoV in Vero E6 cells (Table 3).

Compound 11u appeared so far the best compromise compound, yet for each of the individual viral enzymes, the following compounds proved superior: P2 = cyclopropylmethyl (compound 11s) for SARS-CoV M^pro, P2 = isobutyl (compound 11n) and P2 = cyclopropylmethyl (11s) for HCoV-NL63 M^pro, P2 = benzyl (11a) or cyclohexylmethyl (11r) for EV-A71 3C^pro, and 11r for CVB3 3C^pro. In other words, the nearly equipotent 11u is indeed a compromise. Therefore, in view of the surprisingly good antiviral activity of 11r against HCoV 229E in Huh7 cells, we relaxed the condition that the universal inhibitor should show good activity against the recombinant HCoV-NL63 M^pro and selected 11r (P2 = cyclohexylmethyl) as the lead compound for further development. This compound exhibited submicromolar IC₅₀ values against CVB3 3C^pro and SARS-CoV M^pro and IC₅₀ = 1.7 μM against EV-A71 3C^pro (Table 1), as well as similarly low EC₅₀ values in the replicons of these viruses (Table 2). In Huh7 cells infected with MERS-CoV, the performance of this compound was excellent, with EC₅₀ = 0.0004 μM, and even against HCoV 229E in Huh7 cells and SARS-CoV in Vero E6 cells, EC₅₀ values of 1.8 and 2.1 μM, respectively, were observed (Table 3). Also, in enterovirus-infected cell cultures, the compound performed well, with EC₅₀ values of 0.7 μM or below against HRV2, HRV14, and EV-D68 in HeLa (Rh) cells and selectivity values > 15. The only concern is the activity of the compound against EV-A71 in RD cells, for which the EC₅₀ value was 3.7 μM, resulting in too low a therapeutic index. On the other hand, only weak toxicity was detected for 11r in Vero or Huh-T7 cells. Preliminary pharmacokinetics tests with the compound in mice did not indicate a toxicity problem (to be published elsewhere).

Discussion

We describe here the structure-based design, the synthesis, and the assessment of capped dipeptide α-ketoamides that target the main protease of alpha- or betacoronaviruses as well as the 3C protease of enteroviruses. Through crystallographic analyses of a total of six inhibitor complexes of three different proteases in this study, we found the α-ketoamide warhead (—CO—CO—NH—) to be sterically more versatile than other warheads such as Michael acceptors (—CH═CH—CO—) and aldehydes (—CH═O) because it features two acceptors for hydrogen bonds from the protein, namely, the α-keto oxygen and the amide oxygen, whereas the other warheads have only one such acceptor. In the various complexes, the hydroxy group (or oxyanion) of the thiohemiketal that is formed by the nucleophilic attack of the active-site cysteine residue onto the α-keto carbon can accept one or two hydrogen bonds from the main-chain amides of the oxyanion hole. In addition, the amide oxygen of the inhibitor accepts a hydrogen bond from the catalytic His side chain. Alternatively, the thiohemiketal can interact with the catalytic His residue and the amide oxygen with the main-chain amides of the oxyanion hole. Depending on the exact interaction, the stereochemistry at the thiohemiketal C atom would be different. We have previously observed a similar difference in the case of aldehyde inhibitors, where the single interaction point, the oxyanion of the thiohemiacetal, can accept a hydrogen bond either from the oxyanion hole or from the catalytic His side chain, (37) resulting in different stereochemistry of the thiohemiacetal carbon. Both α-ketoamides and aldehydes react reversibly with the catalytic nucleophile of proteases, whereas Michael acceptors form irreversible adducts.

In addition to better matching the H-bonding donor/acceptor properties of the catalytic center through offering two hydrogen-bond acceptors instead of one, α-ketoamides have another big advantage over aldehydes and α,β-unsaturated esters (Michael acceptors) in that they allow easy extension of the inhibitors to probe the primed specificity subsites beyond S1′, although this has so far rarely been explored (e.g., ref (38) in the case of calpain).

The most prominent α-ketoamide drugs are probably telaprivir and boceprivir, peptidomimetic inhibitors of the HCV NS3/4A protease, (39,40) which have helped revolutionize the treatment of chronic HCV infections. For viral cysteine proteases, α-ketoamides have only occasionally been described as inhibitors, and few systematic studies have been carried out.

A number of capped dipeptidyl α-ketoamides have been described as inhibitors of the norovirus 3C-like protease. (41) These were optimized with respect to their P1′ substituent, whereas P2 was isobutyl in most cases and occasionally benzyl. The former displayed IC₅₀ values 1 order of magnitude lower than the latter, indicating that the S2 pocket of the norovirus 3CL protease is fairly small. Although we did not include the norovirus 3CL^pro in our study, expanding the target range of our inhibitors to norovirus is probably a realistic undertaking.

While our study was underway, Zeng et al. (42) published a series of α-ketoamides as inhibitors of the EV-A71 3C^pro. These authors mainly studied the structure–activity relationships of the P1′ residue and found small alkyl substituents to be superior to larger ones. Interestingly, they also reported that a six-membered δ-lactam in the P1 position led to 2–3 times higher activities, compared to the five-membered γ-lactam. At the same time, Kim et al. (43) described a series of five α-ketoamides with P1′ = cyclopropyl that showed submicromolar activity against EV-D68 and two HRV strains.

Occasionally, individual α-ketoamides have been reported in the literature as inhibitors of both the enterovirus 3C protease and the coronavirus main protease. A single capped dipeptidyl α-ketoamide, Cbz-Leu-Gln-Lactam-CO–CO-NH-iPr, was described, which inhibited the recombinant transmissible gastroenteritis virus (TGEV) and SARS-CoV M^pros as well as human rhinovirus and poliovirus 3C^pros in the one-digit micromolar range. (44) Coded GC-375, this compound showed poor activity in cell culture against EV-A71 though (EC₅₀ = 15.2 μM), probably because P2 was isobutyl. As we have shown here, an isobutyl side chain in the P2 position of the inhibitors is too small to completely fill the S2 pocket of the EV-A71 3C^pro and the CVB3 3C^pro.

Among a series of aldehydes, Prior et al. (45) described the capped tripeptidyl α-ketoamide Cbz-1-naphthylalanine-Leu-Gln-Lactam-CO–CO-NH-iPr, which showed IC₅₀ values in the three-digit nanomolar range against HRV 3C^pro and SARS-CoV M^pro, as well as EC₅₀ values of 0.03 μM against HRV18 and 0.5 μM against HCoV 229E in cell cultures. No optimization of this compound was performed, and no toxicity data have been reported.

For compounds with warheads other than α-ketoamides, in vitro activity against both corona- and enteroviruses has also occasionally been reported. Lee et al. (46) described three peptidyl Michael acceptors that displayed inhibitory activity against the M^pros of SARS-CoV and HCoV 229E as well as against the 3C^pro of CVB3. These inhibitors had an IC₅₀ 10–20 times higher for the CVB3 enzyme, compared to SARS-CoV M^pro. P2 was invariably isobutyl (leucine) in these compounds, suggesting that further improvement might be possible. (47)

In addition to Michael acceptors, peptide aldehydes have also been used to explore the inhibition of coronavirus M^pros as well as enterovirus 3C^pros. Kim et al. (44) reported a dipeptidyl aldehyde and its bisulfite adduct, both of which exhibited good inhibitory activities against the isolated 3C proteases of human rhinovirus and poliovirus as well as against the 3C-like proteases of a number of coronaviruses, but antiviral activities in cell culture against EV-A71 were poor (EC₅₀ > 10 μM), again most probably due to P2 being isobutyl (leucine).

In our series of compounds, we used P1 = Gln-Lactam (γ-lactam) throughout because this substituent has proven to be an excellent surrogate for glutamine. (29,32) While we made some efforts to optimize the P1′ residue of the compounds as well as the N-cap (P3), we mainly focused on optimization of the P2 substituent. In nearly all studies aiming at discovering peptidomimetic inhibitors of coronavirus M^pros, P2 is invariably isobutyl (leucine), and this residue has also been used in the efforts to design compounds that would inhibit enterovirus 3C^pros as well (see above). From crystal structures of our early lead compound, 11a (cinnamoyl-Phe-Gln-Lactam-CO–CO-NH-Bz), in complex with the M^pros of HCoV NL63 (as representative of the alphacoronavirus proteases) and SARS-CoV (beta-CoV) as well as the 3C^pro of Coxsackievirus B3 (enterovirus proteases), we found that the S2 pocket has fundamentally different shapes in these enzymes. In the SARS-CoV M^pro, the S2 subsite is a deep hydrophobic pocket that is truly three-dimensional in shape: the "walls" of the groove are formed by the polypeptide main chain around residues 186–188 as well as by the side chains of His41 (of the catalytic dyad) and Gln189, whereas the "floor" is formed by Met165 and the "lid" by residues 45–51, in particular Met49. The two methionines provide important interaction points for the P2 substituents of inhibitors; while these interactions are mostly hydrophobic in character, we have previously described the surprising observation of the carboxylate of an aspartic residue in P2 that made polar interactions with the sulfur atoms of these methionines. (37) Because the pocket offers so many opportunities for interaction and features a pronounced plasticity, P2 substituents such as isobutyl (from Leu), which are too small to fill the pocket entirely, can still generate sufficient binding enthalpy. Accordingly, the S2 pocket of SARS-CoV M^pro is the most tolerant among the three enzymes investigated here, in terms of versatility of the P2 substituents accepted.

In the S2 pocket of the HCoV-NL63 M^pro, Gln189 is replaced by proline, and this change is accompanied by a significant loss of flexibility; whereas the side chain of Gln189 of SARS-CoV M^pro is found to accommodate its conformation according to the steric requirements of the P2 substituent, the proline is less flexible, leading to a much smaller space at the entrance to the pocket. As a consequence, a P2-benzyl substituent is hindered from penetrating deeply into the pocket, whereas the smaller and more flexible isobutyl group of P2-Leu is not.

Finally, in the 3C^pros of EV-A71 and CVB3, the S2 pocket lacks a lid; i.e., it is open to one side. As a consequence, it offers fewer interaction points for P2 substituents of inhibitors so that such substituents must reach the "back wall" of the pocket (formed by Arg39, Asn69, and Glu71) in order to create sufficient binding energy. Hence, large aromatic substituents such as benzyl are favored by the enterovirus 3C^pros.

When we introduced a fluoro substituent in the para position of the P2-benzyl group of our lead compound, 11a, we observed good activity against the enterovirus 3C^pros but complete inactivity against the coronavirus M^pros (see Table 1, compound 11m). This is easily explained on the basis of the crystal structures: In the enterovirus 3C^pros, the fluorine can accept a hydrogen bond from Arg39 (ref (30)), whereas in the coronavirus M^pros, there would be electrostatic repulsion from the main-chain carbonyls of residues 186 and 188. In agreement with this, rupintrivir (which has P2 = p-fluorobenzyl) is a good inhibitor of the enteroviral 3C^pros, (46) but not of the coronaviral main proteases, as we predicted earlier. (28)

In this structure-based inhibitor optimization study, we achieved major improvements over our original lead compound, 11a, by systematically varying the size and the flexibility of the P2 substituent. The compound presenting so far the best compromise between the different requirements of the S2 pockets (SARS-CoV M^pro, large and covered; HCoV-NL63 M^pro, small and covered; CVB3 3C^pro, large and open) is 11u (P2 = cyclopentylmethyl), which has satisfactory broad-spectrum activity against all proteases tested. However, with regard to its antiviral activities in cell cultures, it is inferior to 11r (P2 = cyclohexylmethyl). The latter compound exhibits very good inhibitory activity against the SARS-CoV M^pro as well as the enterovirus 3C^pro, and its performance in the SARS-CoV and enterovirus replicons is convincing. Being in the low micromolar range (EV-A71, CVB3), the data for the antiviral activity in cell cultures for 11r correlate well with the inhibitory power of the compound against the recombinant proteases as well as in the replicon-based assays. This is not true, though, for the surprisingly good in cellulo activity of 11r against HCoV 229E in Huh7 cells. Also, the correlation does not seem to hold for LLC-MK2 and CaCo2 cells. We tested the antiviral activity of many of our compounds against HCoV NL63 in these two cell types and found that all of them had low or submicromolar EC₅₀ values against this virus in LLC-MK2 cells but were largely inactive in CaCo2 cells (not shown). Furthermore, 11r and all other compounds that we synthesized are inactive (EC₅₀ > 87 μM) against CVB3 in Vero cells (not shown), but exhibit good to excellent activities against the same virus in Huh-T7 cells. We have previously observed similar poor antiviral activities in Vero cells not only for α-ketoamides but also for Michael acceptors (Zhu et al., unpublished work). A similar cell-type dependence is seen for the antiviral activity of 11r against MERS-CoV and SARS-CoV. Whereas the inhibitor exhibits excellent activity against MERS-CoV when Huh7 cells are the host cells (400 pM), the inhibitory activity is weaker by a factor of up to 12,500 when Vero cells are used (EC₅₀ = 5 μM). On the other hand, 11r exhibits excellent anti-MERS-CoV activity in human Calu3 lung cells, i.e., in the primary target cells, where the compound will have to act in a therapeutic setting (A. Kupke, personal communication). As we tested antiviral activity against SARS-CoV exclusively in Vero cells, the EC₅₀ values determined for our compounds against this virus are in the one-digit micromolar range or higher; the best is again compound 11r with EC₅₀ = 2.1 μM. Interestingly, the relatively weaker activity (or even inactivity) of our inhibitors against RNA viruses in Vero cells was observed independently in the virology laboratories in Leuven and in Leiden. It is thus unlikely that the lack of activity in Vero cells is related to problems with the experimental setup. In preliminary experiments, we replaced the P3 cinnamoyl group of 11r by the fluorophor coumaryl and found by fluorescence microscopy that much more inhibitor appeared to accumulate in Huh7 cells compared to Vero cells (D.L., R.H. and Irina Majoul, unpublished).

Regardless of which cell system is the most suitable one for the testing of peptidomimetic antiviral compounds, we next plan to test 11r in small-animal models for MERS and for Coxsackievirus-induced pancreatitis. In parallel, we aim to refine the experiments to quantify the accumulation of peptidomimetic protease inhibitors in different host-cell types in the hope of finding an explanation for the observed cell-type dependencies.

Conclusions

This work demonstrates the power of structure-based approaches in the design of broad-spectrum antiviral compounds with roughly equipotent activity against coronaviruses and enteroviruses. We observed a good correlation between the inhibitory activity of the designed compounds against the isolated proteases, in viral replicons, and in virus-infected Huh7 cells. One of the compounds (11r) exhibits excellent anti-MERS-CoV activity in virus-infected Huh7 cells. Because of the high similarity between the main proteases of SARS-CoV and the novel BetaCoV/Wuhan/2019, we expect 11r to exhibit good antiviral activity against the new coronavirus as well.

Experimental Section

Crystallization and X-ray Structure Determination of Complexes between Viral Proteases and α-Ketoamides

Crystallization

The recombinant production and purification of SARS-CoV M^pro with authentic N and C termini were described in detail previously. (48,49) Using an Amicon YM10 membrane (EMD Millipore), the purified SARS-CoV M^pro was concentrated to 21 mg mL^–1 in buffer A (20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, pH 7.5). Crystallization was performed by equilibrating 1 μL of protein (mixed with 1 μL precipitant solution) against a 500 μL reservoir containing 6–8% polyethylene glycol (PEG) 6,000, 0.1 M MES (pH 6.0), at 20 °C using the vapor diffusion sitting-drop method. Compounds 11a and 11s were dissolved in 100% DMSO at 50 mM and 200 mM stock concentrations, respectively. A crystal of the free enzyme was soaked in cryo-protectant buffer containing 20% MPD, 6% PEG 6,000, 0.1 M MES, 7.5 mM 11a, pH 6.0, for 2 h at 20 °C. Another set of free enzyme crystals was soaked in another cryo-protectant buffer with 6% PEG 6,000, 5% MPD, 0.1 M MES, 15% glycerol, 10 mM 11s, pH 6.0, for 2 h. Subsequently, crystals were fished and flash-cooled in liquid nitrogen prior to data collection.

Crystals of HCoV-NL63 M^pro with 11a were obtained using cocrystallization. The concentrated HCoV-NL63 M^pro (45 mg mL^–1) was incubated with 5 mM 11a for 4 h at 20 °C, followed by setting up crystallization using the vapor diffusion sitting-drop method at 20 °C with equilibration of 1 μL of protein (mixed with 1 μL of mother liquor) against a 500 μL reservoir composed of 0.1 M lithium sulfate monohydrate, 0.1 M sodium citrate tribasic dihydrate, 25% PEG 1,000, pH 6.0. The crystals were protected by a cryo-buffer containing 0.1 M lithium sulfate monohydrate, 0.1 M sodium citrate tribasic dihydrate, 25% PEG 1,000, 15% glycerol, 2 mM 11a, pH 6.0, and flash-cooled in liquid nitrogen.

Crystals of HCoV-NL63 M^pro with 11n or 11f were generated by using the soaking method. Several free-enzyme crystals were soaked in cryo-protectant buffer containing 0.1 M lithium sulfate monohydrate, 0.1 M sodium citrate tribasic dihydrate, 25% PEG 1,000, 15% glycerol, 5 mM 11n (or 11f), pH 6.0. Subsequently, the soaked crystals were flash-cooled in liquid nitrogen.

Freshly prepared CVB3 3C^pro at a concentration of 21.8 mg mL^–1 was incubated with 5 mM 11a predissolved in 100% DMSO at room temperature for 1 h. Some white precipitate appeared in the mixture. Afterward, the sample was centrifuged at 13,000g for 20 min at 4 °C. The supernatant was subjected to crystallization trials using the following, commercially available kits: Sigma (Sigma-Aldrich), Index, and PEG Rx (Hampton Research). Single rod-like crystals were detected both from the Index screen, under the condition of 0.1 M MgCl₂ hexahydrate, 0.1 M Bis-Tris, 25% PEG 3,350, pH 5.5, and from the Sigma screen at 0.2 M Li₂SO₄, 0.1 M Tris-HCl, and 30% PEG 4,000, pH 8.5. Crystal optimization was performed by using the vapor-diffusion sitting-drop method, with 1 μL of the CVB3 3C^pro–inhibitor complex mixed with 1 μL of precipitant solution, and equilibration against a 500 μL reservoir containing 0.1 M Tris-HCl, 0.2 M MgCl₂, pH 8.5, and PEG 3,350 varied from 22% to 27%. Another optimization screen was also performed against a different reservoir, 0.1 M Tris-HCl, 0.2 M MgCl₂, pH range from 7.5 to 8.5, and PEG 4,000 varied from 24% to 34%. Crystals were fished from different drops and protected by cryo-protectant solution consisting of the mother liquor and 10% glycerol. Subsequently, the crystals were flash-cooled with liquid nitrogen.

Diffraction Data Collection, Structure Elucidation, and Refinement

Diffraction data from the crystal of the SARS-CoV M^pro in complex with 11a were collected at 100 K at synchrotron beamline PXI-X06SA (PSI, Villigen, Switzerland) using a Pilatus 6 M detector (DECTRIS). A diffraction data set from the SARS-CoV M^pro crystal with compound 11s was collected at 100 K at beamline P11 of PETRA III (DESY, Hamburg, Germany), using the same type of detector. All diffraction data sets of HCoV-NL63 M^pro complex structures and of the complex of CVB3 3C^pro with 11a were collected at synchrotron beamline BL14.2 of BESSY (Berlin, Germany), using an MX225 CCD detector (Rayonics). All data sets were processed by the program XDSAPP and scaled by SCALA from the CCP4 suite. (50−52) The structure of SARS-CoV M^pro with 11a was determined by molecular replacement with the structure of the complex between SARS-CoV M^pro and SG85 (PDB entry 3TNT; Zhu et al., unpublished) as a search model, employing the MOLREP program (also from the CCP4 suite). (52,53) The complex structures of HCoV-NL63 M^pro with 11a, 11f, and 11n were also determined with MOLREP, using as a search model the structure of the free enzyme determined by us (LZ et al., unpublished). The complex structure between CVB3 3C^pro and 11a was determined based on the search model of the free-enzyme structure (PDB entry 3ZYD; Tan et al., unpublished). Geometric restraints for the compounds 11a, 11f, 11n, and 11s were generated by using JLIGAND (52,54) and built into the F _o – F _c difference density using the COOT software. (55) Refinement of the structures was performed with REFMAC, version 5.8.0131 (refs (52,56,and57)).

Inhibitory Activity Assay of α-Ketoamides

A buffer containing 20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT, pH 7.3, was used for all of the enzymatic assays. Two substrates with the cleavage sites of M^pro and 3C^pro, respectively (indicated by the arrow, ↓), Dabcyl-KTSAVLQ↓SGFRKM-E(Edans)-NH₂ and Dabcyl-KEALFQ↓GPPQF-E(Edans)-NH₂ (95% purity; Biosyntan), were employed in the fluorescence resonance energy transfer (FRET)-based cleavage assay, using a 96-well microtiter plate. The dequenching of the Edans fluorescence due to the cleavage of the substrate as catalyzed by the proteases was monitored at 460 nm with excitation at 360 nm, using an Flx800 fluorescence spectrophotometer (BioTek). Curves of relative fluorescence units (RFU) against substrate concentrations were linear for all substrates up to beyond 50 μM, indicating a minimal influence of the inner-filter effect. Stock solutions of the compounds were prepared by dissolving them in 100% DMSO. The UV absorption of 11a was found to be negligible at λ = 360 nm so that no interference with the FRET signal through the inner-filter effect was to be expected. For the determination of the IC₅₀, different proteases at a specified final concentration (0.5 μM SARS-CoV or HCoV-NL63 M^pro, 2 μM CVB3 3C^pro, 3 μM EV-A71 3C^pro) were separately incubated with the inhibitor at various concentrations (0–100 μM) in reaction buffer at 37 °C for 10 min. Afterward, the reaction was initiated by adding the a FRET peptide substrate at a 20 μM final concentration (final volume: 50 μL). The IC₅₀ value was determined by using the GraphPad Prism 6.0 software (GraphPad). Measurements of enzymatic activity were performed in triplicate and are presented as the mean ± standard deviations (SD).

The assessment of inhibitory activity of α-ketoamides was performed using viral replicons and virus-infected cells

Cells and Viruses

Hepatocellular carcinoma cells (Huh7; ref (58)) and their derivative constitutively expressing T7 RNA polymerase (Huh-T7; ref (59)) were grown in Dulbecco's modified minimal essential medium (DMEM) supplemented with 2 mM glutamine, 100 U mL^–1 penicillin, 100 μg mL^–1 streptomycin sulfate, and fetal calf serum (10% in growth medium and 2% in maintenance medium). Huh-T7 cells were additionally supplemented with Geneticin (G-418 sulfate, 400 μg·mL^–1). Huh-T7 cells were used for the enteroviral replicons as well as for infection experiments with CVB3 strain Nancy.

For enterovirus (except CVB3) infection experiments, human rhabdomyosarcoma cells (RD; for EV-A71; BRCR strain) and HeLa Rh cells (for EV-D68 and human rhinoviruses) were grown in MEM Rega 3 medium supplemented with 1% sodium bicarbonate, 1% l-glutamine, and fetal calf serum (10% in growth medium and 2% in maintenance medium). For HCoV-229E (a kind gift from Volker Thiel (Bern, Switzerland)), culture and infection experiments were carried out as described. (60) For MERS-CoV or SARS-CoV infection experiments, Vero, Vero E6, and Huh7 cells were cultured as described previously. (61,62) Infection of Vero and Huh7 cells with MERS-CoV (strain EMC/2012) and SARS-CoV infection of Vero E6 cells (strain Frankfurt-1) at low multiplicity of infection (MOI) were done as described before. (61,63) All work with live MERS-CoV and SARS-CoV was performed inside biosafety cabinets in biosafety level-3 facilities at the Leiden University Medical Center, The Netherlands.

Viral Replicons

The DNA-launched SARS-CoV replicon harboring Renilla luciferase as reporter directly downstream of the SARS-CoV replicase polyprotein-coding sequence (pp1a, pp1ab, Urbani strain, acc. AY278741), in the context of a bacterial artificial chromosome (BAC) under the control of the CMV promoter, has been described previously (pBAC-REP-RLuc). (33) Apart from the replicase polyprotein, the replicon encodes the following features: the 5′- and 3′-nontranslated regions (NTR), a ribozyme (Rz), the bovine growth hormone sequence, and structural protein N.

Subgenomic replicons of CVB3 (pT7-CVB3-FLuc (34)) and EV-A71 (pT7-EV71-RLuc) harboring T7-controlled complete viral genomes, in which the P1 capsid-coding sequence was replaced by the Firefly (Photinus pyralis) or Renilla (Renilla renifor) luciferase gene, were generously provided by F. van Kuppeveld and B. Zhang, respectively. To prepare CVB3 and EV-A71 replicon RNA transcripts, plasmid DNAs were linearized by digestion with SalI or HindIII (New England Biolabs), respectively. Copy RNA transcripts were synthesized in vitro using linearized DNA templates, T7 RNA polymerase, and the T7 RiboMax Large-Scale RNA Production System (Promega) according to the manufacturer's recommendations.

Transfection

Huh-T7 cells grown in 12-well plates to a confluency of 80%–90% (2–3 × 10⁵ cells/well) were washed with 1 mL of OptiMEM (Invitrogen) and transfected with 0.25 μg of the replication-competent replicon and Lipofectamin2000 or X-tremeGENE9 in 300 μL of OptiMEM (final volume) as recommended by the manufacturer (Invitrogen or Roche, respectively). The transfection mixtures were incubated at 37 °C for 4–5 h (Lipofectamin2000) or overnight (X-tremeGENE9), prior to being replaced with growth medium containing the compound under investigation. For RNA-launched transfection of enteroviral replicons, DMRIE-C was used as a transfection reagent according to the manufacturer's recommendations (Invitrogen). All experiments were done in triplicate or quadruplicate, and the results are presented as mean values ± SD.

Testing for Inhibitory Activity of Candidate Compounds

Initially, we performed a quick assessment of the inhibitory activity of the candidate compounds toward the enteroviral and coronaviral replicons at a concentration of 40 μM in Huh-T7 cells. Compounds that were relatively powerful and nontoxic at this concentration were assayed in a dose-dependent manner to estimate their half-maximal effective concentration (EC₅₀) as well as their cytotoxicity (CC₅₀), as described. (29) In brief, different concentrations of α-ketoamides (40 μM in screening experiments or increasing concentrations (0, 1.25, 2.5, 5, 10, 20, 40 μM) when determining the EC₅₀) were added to growth medium of replicon-transfected Huh-T7 cells. Twenty-four hours later, the cells were washed with 1 mL of phosphate-buffered saline (PBS or OPTIMEM, Invitrogen) and lysed in 0.15 mL of Passive lysis buffer (Promega) at room temperature (RT) for 10 min. After freezing (−80 °C) and thawing (RT), the cell debris was removed by centrifugation (16,000g, 1 min), and the supernatant (10 or 20 μL) was assayed for Firefly or Renilla luciferase activity (Promega or Biotrend Chemikalien) using an Anthos Lucy-3 luminescence plate reader (Anthos Microsystem).

Antiviral Assay with Infectious Enteroviruses

The antiviral activity of the compounds was evaluated in a cytopathic effect (CPE) read-out assay using the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, inner-salt]-based assay. Briefly, 24 h prior to infection, cells were seeded in 96-well plates at a density of 2.5 × 10⁴ (RD cells) or of 1.7 × 10⁴ (HeLa Rh) per well in medium supplemented with 2% FCS. For HRV2 and HRV14 infection, the medium contained 30 mM MgCl₂. The next day, serial dilutions of the compounds and virus inoculum were added. The read-out was performed 3 days post infection as follows: The medium was removed, and 100 μL of 5% MTS in phenol Red-free MEM was added to each well. Plates were incubated for 1 h at 37 °C, and then the optical density at 498 nm (OD₄₉₈) of each well was measured by a microtiter plate reader (Saffire², Tecan). The OD values were converted to the percentage of controls, and the EC₅₀ was calculated by logarithmic interpolation as the concentration of the compound that results in a 50% protective effect against virus-induced CPE. For each condition, cell morphology was also evaluated microscopically.

Antiviral Assays with SARS and MERS Coronaviruses

Assays with MERS-CoV and SARS-CoV were performed as previously described. (61,63) In brief, Huh7, Vero, or Vero E6 cells were seeded in 96-well plates at a density of 1 × 10⁴ (Huh7 and Vero E6) or 2 × 10⁴ cells (Vero) per well. After overnight growth, cells were treated with the indicated compound concentrations or DMSO (solvent control) and infected with an MOI of 0.005 (final volume 150 μL/well in Eagle's minimal essential medium (EMEM) containing 2% FCS, 2 mM l-glutamine, and antibiotics). Huh7 cells were incubated for 2 days and Vero/VeroE6 cells for 3 days, and differences in cell viability caused by virus-induced CPE or by compound-specific side effects were analyzed using the CellTiter 96 AQ_ueous Non-Radioactive Cell Proliferation Assay (Promega), according to the manufacturer's instructions. Absorbance at 490 nm (A ₄₉₀) was measured using a Berthold Mithras LB 940 96-well plate reader (Berthold). Cytotoxic effects caused by compound treatment alone were monitored in parallel plates containing mock-infected cells.

Antiviral Assay with Human Coronavirus 229E

For HCoV-229E infection experiments, 5 × 10⁴ Huh7 cells were infected in triplicate in 24-well plates in 100 μL of DMEM at 0.1 pfu/mL. After 1.5 h of incubation at 37 °C, virus inocula were removed. Cells were washed with DMEM, and complete DMEM (10% FCS, 1% Pen./Strep.) containing the desired concentration of inhibitors (0, 1, 2.5, 5, 10, 20, and 40 μM) was added. After 48 h, the supernatant was collected. Viral RNA was isolated using the Bioline ISOLATE II RNA Mini Kit (no. BIO-52072) according to the manufacturer's instructions and eluted in 30 μL of RNase-free water. qPCR was performed using the Bioline SensiFAST Probe Hi-ROX One-Step Kit (no. BIO-77001) in a Roche Light Cycler96. cDNA was synthesized at 48 °C for 1800 s and 95 °C for 600 s, followed by 45 cycles at 95 °C for 15 s and 60 °C for 60 s at a temperature ramp of 4.4 °C/sec. qPCR primer sequences (adapted from ref (64)) were as follows: 229E-For, 5′-CTACAGATAGAAAAGTTGCTTT-3′; HCoV-229E-Rev, 5′-ggTCGTTTAGTTGAGAAAAGT-3′; 229E-ZNA probe, 5′-6-Fam-AGA (pdC)TT(pdU)G(pdU)GT(pdC)TA(pdC)T-ZNA-3-BHQ-1–3′ (Metabion). Standard curves were prepared using serial dilutions of RNA isolated from virus stock. Data were analyzed using GraphPad Prism 5.0; EC₅₀ values were calculated based on a four-parameter logistic statistics equation. In parallel to the qPCR assays with inhibitors, cell viability assays were performed using the AlamarBlue Cell Viability Reagent (ThermoFisher) according to the manufacturer's instruction. CC₅₀ values were calculated using an inhibitor versus normalized response statistics equation by including proper controls (no inhibitor and 1% Triton-X-100-treated cells).

Determination of the Cell Toxicity of Candidate Compounds

The CellTiter 96Aqueous One Solution Cell Proliferation Assay (MTS test, Promega), the CellTiter Glo assay kit (Promega), the Non-Destructive Cytotoxicity Bio-Assay (ToxiLight (measuring the release of adenylate kinase from damaged cells), Lonza Rockland), or the AlamarBlue Cell Viability Reagent (ThermoFisher) were used to determine the cytotoxic effect of compounds toward host cells according to the manufacturers' recommendations. (29,65)

Chemical Synthesis of α-Ketoamides

General Procedure

Reagents were purchased from commercial sources and used without purification. HSGF 254 (0.15–0.2 mm thickness) was used for analytical thin-layer chromatography (TLC). All products were characterized by their NMR and MS spectra. ¹H NMR spectra were recorded on 300 MHz, 400 MHz, or 500 MHz instruments. Chemical shifts are reported in parts per million (ppm, δ) downfield from tetramethylsilane. Proton coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Mass spectra were recorded using a Bruker ESI ion-trap HCT Ultra. HPLC spectra were recorded by LC20A or LC10A (Shimadzu Corporation) with Shim-pack GIST C18 (5 μm, 4.6 × 150 mm) with three solvent systems (methanol/water, methanol/0.1% HCOOH in water or methanol/0.1% ammonia in water). The purity was determined by reversed-phase HPLC and was ≥95% for all compounds tested biologically.

Synthesis of (2S,4R)-Dimethyl 2-(tert-butoxycarbonylamino)-4-(cyanomethyl)pentanedioate (1)

To a solution of N-Boc-l-glutamic acid dimethyl ester (6.0 g, 21.8 mmol) in THF (60 mL) was added dropwise a solution of lithium bis(trimethylsilyl)amide (LHMDS) in THF (47 mL, 1 M) at −78 °C under nitrogen. The resulting dark mixture was stirred at −78 °C. Meanwhile, bromoacetonitrile (1.62 mL, 23.3 mmol) was added dropwise to the dianion solution over a period of 1 h, while keeping the temperature below −70 °C. The reaction mixture was stirred at −78 °C for an additional 2 h. After the consumption of the reactant was confirmed by TLC analysis, the reaction was quenched by methanol (3 mL), and acetic acid (3 mL) in precooled THF (20 mL) was added. After stirring for 30 min, the cooling bath was removed. The reaction mixture was allowed to warm up to room temperature and then poured into brine (40 mL). The organic layer was concentrated and purified by flash column chromatography (petroleum ether/ethyl acetate = 4/1) to give product 1 (4.92 g, 72%) as a colorless oil. ¹H NMR (CDCl₃, 400 MHz): δ 5.23 (1H, d, J = 9.0 Hz), 4.43–4.36 (1H, m), 3.77(1H, s), 3.76 (1H, s), 2.89–2.69 (3H, m), 2.20–2.14 (2H, m), 1.45 (9H, s). ESI-MS (m/z): 315 (M + H)⁺.

Synthesis of (S)-Methyl 2-(tert-butoxycarbonylamino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (2)

In a hydrogenation flask were placed compound 1 (4.0 g, 12.7 mmol), 5 mL of chloroform, and 60 mL of methanol before the addition of PtO₂. The resulting mixture was stirred under hydrogen at 20 °C for 12 h. Then the mixture was filtered over Celite to remove the catalyst. NaOAc (6.77 g, 25.5 mmol) was added to the filtrate before the resulting mixture was stirred at 60 °C for 12 h. The reaction was quenched with water (30 mL). The suspension was extracted with ethyl acetate. The organic layers were combined, dried (MgSO₄), and filtered. The light-brown filtrate was concentrated and purified by silica gel column chromatography (petroleum ether/ethyl acetate = 4/1) to give the product 2 (2.20 g, 61%) as a white solid. ¹H NMR (CDCl₃): δ 6.02 (1H, br), 5.49 (1H, d, J = 7.8 Hz), 4.27–4.33 (1H, m), 3.72 (3H, s), 3.31–3.36 (2H, m), 2.40–2.49 (2H, m), 2.06–2.16 (1H, m), 1.77–1.89 (2H, m), 1.41 (9H, s). ESI-MS (m/z): 287 (M + H)⁺.

Synthesis of (S)-Methyl 2-Amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate (3)

Compound 2 (1.0 g, 3.5 mmol) was dissolved in 10 mL of dichloromethane (DCM), and then 10 mL of trifluoroacetic acid (TFA) was added. The reaction mixture was stirred at 20 °C for 0.5 h and concentrated in vacuo to get a colorless oil, which could be used for the following step without purification.

ESI-MS (m/z): 187 (M + H)⁺.

Synthesis of Methyl N-Substituted Amino-acid Esters 4

General Procedure

The methyl amino-acid ester hydrochloride (6.0 mmol) was dissolved in 20 mL of CH₂Cl₂, and then acyl chloride (6.0 mmol) and triethylamine (1.69 mL, 12.0 mmol) were added, before the reaction was stirred for 2 h at 20 °C. The reaction mixture was diluted with 20 mL of CH₂Cl₂, washed with 50 mL of saturated brine (2 × 25 mL), and dried over Na₂SO₄. The solvent was evaporated, and product 4 was obtained as a white solid (70–95% yield), which could be used for the next step without further purification.

(S)-Methyl 2-Cinnamamido-3-phenylpropanoate (4a)

Methyl l-phenylalaninate hydrochloride (1.30 g, 6.0 mmol) was dissolved in 20 mL of CH₂Cl₂, and then cinnamoyl chloride (1.00 g, 6.0 mmol) and triethylamine (1.69 mL, 12.0 mmol) were added, before the reaction was stirred for 2 h at room temperature. The reaction mixture was diluted with 20 mL of CH₂Cl₂, washed with 50 mL of saturated brine (2 × 25 mL), and dried over Na₂SO₄. The solvent was evaporated, and the product 4a was obtained as a white solid (1.75 g, 95%), which could be used for the next step without further purification.

Synthesis of N-Substituted Amino Acids 5 (General Procedure)

One M NaOH (5 mL) was added to a solution of compound 4 (3.0 mmol) in methanol (5 mL). The reaction was stirred for 20 min at 20 °C. Then 1 M HCl was added to the reaction solution until pH = 1. Then the reaction mixture was extracted with 100 mL of CH₂Cl₂ (2 × 50 mL), and the organic layer was washed with 50 mL of brine and dried over Na₂SO₄. The solvent was evaporated and the crude material purified on silica and eluted with mixtures of CH₂Cl₂/MeOH (20/1) to afford the product 5 (90–96% yield) as a white solid.

Synthesis of Compounds 6 (General Procedure)

Compound 5 (2.7 mmol) was dissolved in 10 mL of dry CH₂Cl₂. To this solution, 1.5 equiv (1.54 g) of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) was added, and the reaction was stirred for 0.5 h at 20 °C. Then compound 3 (500 mg, 2.7 mmol) and TEA (0.70 mL, 5.42 mmol) were added to the reaction. The reaction was stirred for another 6 h. The reaction mixture was poured into 10 mL of water. The aqueous solution was extracted with 50 mL of CH₂Cl₂ (2 × 25 mL) and washed with 50 mL of saturated brine (2 × 25 mL) and dried over Na₂SO₄. The solvent was evaporated and the crude material purified on silica and eluted with a mixture of CH₂Cl₂/MeOH (40/1) to give the product 6 (62–84% yield).

Synthesis of Alcohols 7 (General Procedure)

Compound 6 (1.1 mmol) was dissolved in methanol (40 mL), and then NaBH₄ (0.34 g, 8.8 mmol) was added under ambient conditions. The reaction mixture was stirred at 20 °C for 2 h. Then the reaction was quenched with water (30 mL). The suspension was extracted with ethyl acetate. The organic layers were combined, dried, and filtered. The filtrate was evaporated to dryness and could be used for the next step without further purification (46–85% yield).

Synthesis of Aldehydes 8 (General Procedure)

Compound 7 (0.75 mmol) was dissolved in CH₂Cl₂, and then Dess–Martin periodinane (337 mg, 0.79 mmol) and NaHCO₃ (66 mg, 0.79 mmol) were added. The resulting mixture was stirred at 20 °C for 1 h. The mixture was concentrated and purified by column chromatography on silica gel (CH₂Cl₂/MeOH = 20/1) to give the product 8 as a white solid (88–95% yield).

Synthesis of Compounds 9 (General Procedure)

Compound 8 (0.40 mmol) was dissolved in CH₂Cl₂, and then acetic acid (0.028 g, 0.47 mmol) and isocyanide (0.43 mmol) were added successively to the solution. The reaction was stirred at 20 °C for 24 h. Then the solvent was evaporated and the crude material purified on silica and eluted with a mixture of CH₂Cl₂/MeOH (20/1) to give the product 9 (46–84%).

Synthesis of α-Hydroxyamides 10 (General Procedure)

One M NaOH (0.5 mL) was added to a solution of compound 9 (0.164 mmol) in methanol (5 mL). The reaction was stirred at 20 °C for 0.5 h until the consumption of compound 9 was confirmed by TLC analysis. Then, 1 M HCl was added to the reaction solution until pH = 7. Following this, the solvent was evaporated to generate the product 10 as a white solid, which could be used directly in the next step.

Synthesis of α-Ketoamides 11 (General Procedure)

Compound 10 was dissolved in CH₂Cl₂, and then Dess–Martin periodinane (74 mg, 0.176 mmol) and NaHCO₃ (30 mg, 0.176 mmol) were added. The resulting mixture was stirred at 20 °C for 1 h. The mixture was concentrated and purified by column chromatography on silica gel (CH₂Cl₂/MeOH = 20/1) to give the α-ketoamides 11 as a light yellow solid (52–79% in two steps).

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-phenylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11a):

75% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.92 (d, J = 7.6 Hz, 1H), 7.56 (d, J = 15.2 Hz, 1H), 7.45–7.43 (m, 2H), 7.35–7.19 (m, 12H), 7.02–6.98 (m, 1H), 6.48 (d, J = 15.2 Hz, 1H), 6.44–6.42 (m, 1H), 5.01–4.92 (m, 2H), 4.46 (d, J = 8.4 Hz, 2H), 3.25–3.03 (m, 4H), 2.24–2.21 (m, 2H), 1.95–1.86 (m, 1H), 1.74–1.69 (m, 1H), 1.55–1.49 (m, 1H) ppm. ESI-MS (m/z): 567 [M + H]⁺.

tert-Butyl ((S)-4-(Benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)carbamate (11f)

¹H NMR (300 MHz, CDCl₃): δ 7.32–7.22 (m, 5H), 6.47 (br, 1H), 5.10 (d, J = 8.4 Hz, 1H), 4.37–4.26 (m, 3H), 3.37–3.32 (m, 2H), 2.53–2.47 (m, 2H), 2.05–1.98 (m, 1H), 1.85–1.79 (m, 1H), 1.62–1.56 (m, 1H), 1.44 (s, 9H) ppm. ESI-MS (m/z): 390 [M + H]⁺.

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-(4-fluorophenyl)propanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11m):

78% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.91 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 15.2 Hz, 1H), 7.43–7.40 (m, 2H), 7.35–7.11 (m, 11H), 7.01–6.98 (m, 1H), 6.46 (d, J = 15.2 Hz, 1H), 6.35–6.31 (m, 1H), 4.99–4.91 (m, 2H), 4.43 (d, J = 8.8 Hz, 2H), 3.27–3.12 (m, 3H), 3.05–2.99 (m, 1H), 2.24–2.21 (m, 2H), 2.03–1.96 (m, 1H), 1.72–1.54 (m, 2H) ppm. ESI-MS (m/z): 585 [M + H]⁺.

(S)-N-((S)-4-(Benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)-2-cinnamamido-4-methylpentanamide (11n):

57% yield. ¹H NMR (400 MHz, CDCl₃): δ 8.01 (d, J = 7.6 Hz, 1H), 7.58 (d, J = 15.6 Hz, 1H), 7.43–7.35 (m, 5H), 7.33–7.14 (m, 4H), 7.02–6.98 (m, 1H), 6.48 (d, J = 15.6 Hz, 1H), 6.37–6.32 (m, 1H), 4.94–4.86 (m, 1H), 4.68–4.62 (m, 1H), 4.46 (d, J = 8.4 Hz, 2H), 3.25–3.11(m, 1H), 3.09–3.06 (m, 1H), 2.25–2.21 (m, 2H), 1.99–1.92 (m, 1H), 1.73–1.64 (m, 3H), 1.58–1.48 (m, 2H), 0.92 (d, J = 8.4 Hz, 3H), 0.88 (d, J = 8.4 Hz, 3H) ppm. ESI-MS (m/z): 533 [M + H]⁺.

(S)-N-((S)-4-(Benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)-2-cinnamamidohexanamide (11o):

76% yield. ¹H NMR (400 MHz, CDCl₃): δ 7.91 (d, J = 7.6 Hz, 1H), 7.55 (d, J = 15.2 Hz, 1H), 7.43–7.36 (m, 5H), 7.28–7.14 (m, 4H), 7.01–6.98 (m, 1H), 6.45 (d, J = 15.2 Hz, 1H), 6.37–6.32 (m, 1H), 4.98–4.91 (m, 1H), 4.73–4.67 (m, 1H), 4.48 (d, J = 8.0 Hz, 2H), 3.25–3.11(m, 1H), 3.09–3.03 (m, 1H), 2.25–2.21 (m, 2H), 1.92–1.86 (m, 1H), 1.73–1.64 (m, 3H), 1.56–1.52 (m, 1H), 1.36–1.25 (m, 4H), 0.93 (t, J = 8.4 Hz, 3H) ppm. ESI-MS (m/z): 533 [M + H]⁺.

(S)-N-((S)-4-(Benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)-2-cinnamamidopent-4-ynamide (11p):

65% yield. ¹H NMR (500 MHz, CDCl₃): δ 8.56 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 15.0 Hz, 1H), 7.53–7.46 (m, 2H), 7.38–7.17 (m, 7H), 6.53–6.42 (m, 2H), 5.32–5.25 (m, 1H), 4.85–4.65 (m, 1H), 4.47 (d, J = 8.5 Hz, 2H), 3.43–3.29 (m, 3H), 2.59–2.45 (m, 1H), 2.20–1.60 (m, 7H) ppm. ESI-MS (m/z): 515 [M + H]⁺.

(S)-N-Benzyl-3-((S)-2-cinnamamido-2-cyclopropylacetamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11q):

66% yield. ¹H NMR (500 MHz, CDCl₃): δ 8.62 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 15.0 Hz, 1H), 7.53–7.43 (m, 2H), 7.35–7.17 (m, 7H), 6.76–6.69 (m, 1H), 6.59–6.48 (m, 1H), 5.35–5.25 (m, 1H), 4.85–4.72 (m, 1H), 4.48 (d, J = 8.5 Hz, 2H), 3.38–3.22 (m, 2H), 2.62–2.45 (m, 1H), 2.12–1.63 (m, 4H), 1.20–0.92 (m, 1H), 0.46 (t, J = 7.0 Hz, 2H), 0.16–0.07 (m, 2H) ppm. ESI-MS (m/z): 517 [M + H]⁺.

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-cyclohexylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11r):

71% yield. ¹H NMR (500 MHz, CDCl₃): δ 8.56 (t, J = 6.0 Hz, 1H), 7.61 (d, J = 16.0 Hz, 1H), 7.52–7.44 (m, 3H), 7.35–7.20 (m, 6H), 6.66–6.59 (m, 1H), 6.48 (d, J = 13.0 Hz, 1H), 5.32–5.27 (m, 1H), 4.95–4.75 (m, 1H), 4.48 (d, J = 6.5 Hz, 2H), 3.39–3.29 (m, 2H), 2.65–2.35 (m, 2H), 2.09–1.68 (m, 10H), 1.29–1.16 (m, 4H), 1.00–0.88 (m, 2H) ppm. ESI-MS (m/z): 573 [M + H]⁺.

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-cyclopropylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11s):

64% yield. ¹H NMR (500 MHz, CDCl₃): δ 8.64 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 15.0 Hz, 1H), 7.52–7.46 (m, 2H), 7.36–7.17 (m, 7H), 6.54–6.42 (m, 2H), 5.35–5.25 (m, 1H), 4.85–4.75 (m, 1H), 4.46 (d, J = 8.5 Hz, 2H), 3.38–3.29 (m, 2H), 2.65–2.35 (m, 1H), 2.15–1.90 (m, 2H), 1.85–1.60 (m, 4H), 0.90–0.72 (m, 1H), 0.47 (t, J = 7.0 Hz, 2H), 0.15–0.07 (m, 2H) ppm. ESI-MS (m/z): 531 [M + H]⁺.

(S)-N-benzyl-3-((S)-2-cinnamamido-3-cyclobutylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11t):

77% yield. ¹H NMR (500 MHz, CDCl₃): δ 8.62 (t, J = 6.5 Hz, 1H), 7.60 (d, J = 15.0 Hz, 1H), 7.52–7.46 (m, 2H), 7.35–7.19 (m, 7H), 6.72–6.60 (m, 1H), 6.48 (d, J = 15.0 Hz, 1H), 5.32–5.26 (m, 1H), 4.77–4.69 (m, 1H), 4.49 (d, J = 6.5 Hz, 2H), 3.40–3.31 (m, 2H), 2.60–2.35 (m, 3H), 2.09–1.68 (m, 11H) ppm. ESI-MS (m/z): 545 [M + H]⁺.

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-cyclopentylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11u):

79% yield. ¹H NMR (500 MHz, CDCl₃): δ 7.60 (d, J = 15.0 Hz, 1H), 7.50–7.44 (m, 2H), 7.36–7.20 (m, 7H), 6.76–6.69 (m, 1H), 6.59–6.48 (m, 1H), 5.35–5.27 (m, 1H), 4.95–4.65 (m, 1H), 4.45 (d, J = 6.5 Hz, 2H), 3.38–3.29 (m, 2H), 2.65–2.35 (m, 1H), 2.00–1.38 (m, 13H), 1.20–1.00 (m, 2H) ppm. ESI-MS (m/z): 559 [M + H]⁺.

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01828.

Detailed results of the variation of the P1′ and P3 substituents; synthesis of α-ketoamides 11b–11e and 11g–11l; inhibitory activities (IC₅₀ (μM)) of α-ketoamides with P1′ and P3 modifications against viral proteases; crystallographic data for complexes between viral proteases and α-ketoamides (PDF)
Molecular formula strings and biological data (CSV)

jm9b01828_si_001.pdf (459.88 kb)
jm9b01828_si_002.csv (4.56 kb)

Atomic coordinates include SARS-CoV M^pro in complex with compounds 11a (5N19), 11s (5N5O), HCoV-NL63 M^pro in complex with 11a (6FV2), 11n (6FV1), 11f (5NH0), and CVB3 3C^pro in complex with 11a (5NFS).

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Author Information

- Rolf Hilgenfeld - Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562 Lübeck, Germany; German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems Site, University of Lübeck, 23562 Lübeck, Germany; Shanghai Institute of Materia Medica, 201203 Shanghai, China; http://orcid.org/0000-0001-8850-2977; Email: [email protected]
- Linlin Zhang - Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562 Lübeck, Germany; German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems Site, University of Lübeck, 23562 Lübeck, Germany
- Daizong Lin - Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562 Lübeck, Germany; German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel-Riems Site, University of Lübeck, 23562 Lübeck, Germany; Shanghai Institute of Materia Medica, 201203 Shanghai, China; Present Address: D.L.: Changchun Discovery Sciences Ltd., Changchun 130012, China
- Yuri Kusov - Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562 Lübeck, Germany
- Yong Nian - Shanghai Institute of Materia Medica, 201203 Shanghai, China
- Qingjun Ma - Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, 23562 Lübeck, Germany
- Jiang Wang - Shanghai Institute of Materia Medica, 201203 Shanghai, China
- Albrecht von Brunn - Max von Pettenkofer Institute, Ludwig-Maximilians-University Munich, 80336 Munich, Germany
- Pieter Leyssen - Rega Institute for Medical Research, University of Leuven, 3000 Leuven, Belgium
- Kristina Lanko - Rega Institute for Medical Research, University of Leuven, 3000 Leuven, Belgium
- Johan Neyts - Rega Institute for Medical Research, University of Leuven, 3000 Leuven, Belgium
- Adriaan de Wilde - Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
- Eric J. Snijder - Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
L.Z. and D.L. contributed equally. The manuscript was written through contributions by all authors. All authors have given approval to the final version of the manuscript.
Financial support by the European Commission through its SILVER project (contract HEALTH-F3-2010-260644 with R.H., J.N., and E.J.S.) and the German Center for Infection Research (DZIF; TTU 01.803 to R.H. and A.v.B.) is gratefully acknowledged. H.L. thanks the Natural Science Foundation of China (81620108027) for support.
The authors declare no competing financial interest.

Acknowledgments

We thank Doris Mutschall, Javier Carbajo-Lozoya, Dev Raj Bairad, and Sebastian Schwinghammer for expert technical assistance, Dr. Bo Zhang and Prof. Frank van Kuppeveld (Wuhan and Utrecht, respectively) for the EV-A71 and CVB3 replicons, Prof. Ron Fouchier (Rotterdam) for the EMC/2012 strain of MERS-CoV, and Prof. Volker Thiel (Berne) for HCoV 229E. We are grateful to Dr. Naoki Sakai and the staff at synchrotron beamlines for help with diffraction data collection.

Abbreviations Used
3CL^pro	3C-like protease
3C^pro	3C protease
A ₄₉₀	absorbance at 490 nm
ap	antiperiplanar
BAC	bacterial artificial chromosome
CPE	cytopathic effect
CVA16	Coxsackievirus A16
CVB3	Coxsackievirus B3
DMEM	Dulbecco's modified minimal essential medium
EMEM	Eagle's minimal essential medium
EV	enterovirus
FIPV	Feline Infectious Peritonitis Virus
FRET	fluorescence resonance energy transfer
Gln-Lactam	glutamine lactam
HCoV	human coronavirus
HFMD	hand, foot, and mouth disease
HRV	human rhinovirus
MERS-CoV	Middle-East respiratory syndrome coronavirus
M^pro	main protease
Nsp5	nonstructural protein 5
NTR	nontranslated region
OD₄₉₈	optical density at 498 nm
PEG	polyethylene glycol
RFU	relative fluorescence units
Rz	ribozyme
SARS	severe acute respiratory syndrome
SARS-CoV	SARS coronavirus
-sc	(−)-synclinal
SD	standard deviation
TLC	thin-layer chromatography

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We investigated the clinical impact of human coronaviruses (HCoV) OC43, 229E, HKU1 and NL63 in pediatric patients with cystic fibrosis (CF) during routine and exacerbation visits. A total of 408 nasopharyngeal aspirate samples were obtained from 103 patients over a 1-year period. Samples positive for HCoV were submitted for nucleotide sequencing to determine the species. Nineteen samples (4.65%) were positive for HCoV, of which 8 were positive for NL63, 6 for OC43, 4 for HKU1, and 1 for 229E. Identification of HCoV was not associated with an increased rate of respiratory exacerbations, but NL63-positive patients had higher exacerbation rates than patients who were positive for other HCoV species.

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Mayer, K. ; Nellessen, C. ; Hahn-Ast, C. ; Schumacher, M. ; Pietzonka, S. ; Eis-Hubinger, A. M. ; Drosten, C. ; Brossart, P. ; Wolf, D. Fatal outcome of human coronavirus NL63 infection despite successful viral elimination by IFN-alpha in a patient with newly diagnosed ALL. Eur. J. Haematol. 2016, 97 , 208– 210, DOI: 10.1111/ejh.12744

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Mayer Karin; Nellessen Cordula; Hahn-Ast Corinna; Schumacher Martin; Brossart Peter; Wolf Dominik; Pietzonka Sandra; Eis-Hubinger Anna M; Drosten Christian

European journal of haematology (2016), 97 (2), 208-210 ISSN:.

INTRODUCTION: Human coronavirus NL63 (HCoV-NL63) is one of four common human respiratory coronaviruses. Despite high incidence, HCoV infections are grossly understudied. We here report a case of HCoV infection in a leukemia patient with fatal ARDS despite successful virus elimination by pegylated interferon-alpha (PEG-IFN-α). CASE: The 27-year-old female pre-T-ALL patient was treated according to the German-Multicenter Trial for Adult ALL protocol. No relevant infectious complications were seen until day 35 when the neutropenic patient developed fever without any clinical focus. Antibiotic prophylaxis was switched to meropenem. The patient deteriorated rapidly with respiratory failure due to ARDS. Anti-infectious therapy was escalated to additional linezolid and liposomal amphotericine-B. BAL revealed a significant viral load of HCoV-NL63. Based on successful application of PEG-IFN against the related SARS coronavirus in animal experiments, a single injection of 180 μg PEG-IFN-α2b was applied. Despite immediate initiation of treatment and elimination of virus in subsequent tests, the progressive lung failure led to death 7 d after onset of fever with massive lung bleeding as a consequence of diffuse alveolar hemorrhage. CONCLUSION: This report emphasizes the fatal consequences of common respiratory virus infections in immunocompromised patients. HCoV-NL63 replication can be reduced by treatment with peg-IFN if diagnosed early.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC28nosFWkug%253D%253D&md5=76da5928b496e9255890f49137a31f6a
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Lee, J. ; Storch, G. A. Characterization of human coronavirus OC43 and human coronavirus NL63 infections among hospitalized children < 5 years of age. Pediatr. Infect. Dis. J. 2014, 33 , 814– 820, DOI: 10.1097/INF.0000000000000292

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Characterization of human coronavirus OC43 and human coronavirus NL63 infections among hospitalized children <5 years of age

Lee Jina; Storch Gregory A

The Pediatric infectious disease journal (2014), 33 (8), 814-20 ISSN:.

BACKGROUND: Multiplex molecular assays now make it possible for clinical laboratories to detect human coronaviruses (HCoVs). We investigated the clinical characteristics of HCoV-OC43 and HCoV-NL63 in patients <5 years of age during a recent coronavirus season. METHODS: Respiratory viruses were detected using a multiplex molecular assay at St. Louis Childrens Hospital starting in November 2012. We analyzed demographic and clinical data from all patients <5 years of age with solo detection of HCoV-OC43 (n = 52) and HCoV-NL63 (n = 44) and for comparison, samples of children with respiratory syncytial virus, parainfluenza virus and picornaviruses. RESULTS: During the study period, HCoV-OC43 (4%) was the 5th and HCoV-NL63 the 8th (2%) most common respiratory virus. Coinfections were detected in 35% and 38% of children with HCoV-OC43 and HCoV-NL63, respectively. Croup was more common with HCoV-NL63 (30%) than with HCoV-OC43 (2%). Lower respiratory tract infection occurred in 33% of children with HCoV-OC43 and 25% of children with HCoV-NL63. Severe illness was less common in HCoV-NL63, HCoV-OC43 and parainfluenza virus (14%, each) compared with respiratory syncytial virus (30%) and picornaviruses (26%; P = 0.055 for HCoVs combined compared with the other respiratory viruses) and occurred mainly in those with underlying medical conditions. CONCLUSIONS: Infections caused by HCoV-OC43 and HCoV-NL63 are common and include some with lower respiratory tract involvement and severe disease, especially in children with underlying medical conditions. Overall, a substantial burden of disease associated with both HCoV-OC43 and HCoV-NL63 was observed for hospitalized children <5 years of age.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cvoslemsw%253D%253D&md5=cccdb0eeb65c613fe7fcdc06cc71a337
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Oermann, C. M. ; Schuster, J. E. ; Conners, G. P. ; Newland, J. G. ; Selvarangan, R. ; Jackson, M. A. Enterovirus D68. A focused review and clinical highlights from the 2014 U.S. outbreak. Ann. Am. Thorac. Soc. 2015, 12 , 775– 781, DOI: 10.1513/AnnalsATS.201412-592FR

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Enterovirus d68. A focused review and clinical highlights from the 2014 U.S. Outbreak

Oermann Christopher M; Schuster Jennifer E; Conners Gregory P; Newland Jason G; Selvarangan Rangaraj; Jackson Mary Anne

Annals of the American Thoracic Society (2015), 12 (5), 775-81 ISSN:.

Enterovirus D68 (EV-D68), a member of the Picornaviridae family, was first identified in 1962 and is part of a group of small, nonenveloped RNA viruses. As a family, these viruses are among the most common causes of disease among humans. However, outbreaks of disease attributable to EV-D68 have been rarely reported in the previous 4 decades. Reports from a few localized outbreaks since 2008 describe severe lower respiratory tract infection in children. In the late summer of 2014, EV-D68 caused a geographically widespread outbreak of respiratory disease of unprecedented magnitude in the United States. The Centers for Disease Control and Prevention was first notified of increased respiratory viral activity by Children's Mercy Hospitals (CMH) in Kansas City, Missouri, and EV-D68 was identified in 50% of nasopharyngeal specimens initially tested. Between mid-August and December 18, 2014, confirmed cases of lower respiratory tract infection caused by EV-D68 were reported in 1,152 people in 49 states and the District of Columbia. A focused review of EV-D68 respiratory disease and clinical highlights from the 2014 U.S. outbreak are presented here.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MrosFOjug%253D%253D&md5=5f40acd61e68da8e47fdaf18a3959ee6
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Xing, W. ; Liao, Q. ; Viboud, C. ; Zhang, J. ; Sun, J. ; Wu, J. T. ; Chang, Z. ; Liu, F. ; Fang, V. J. ; Zheng, Y. ; Cowling, B. J. ; Varma, J. K. ; Farrar, J. J. ; Leung, G. M. ; Yu, H. Hand, foot, and mouth disease in China, 2008–12: an epidemiological study. Lancet Infect. Dis. 2014, 14 , 308– 318, DOI: 10.1016/S1473-3099(13)70342-6

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Hand, foot, and mouth disease in China, 2008-12: an epidemiological study

Xing Weijia; Liao Qiaohong; Viboud Cecile; Zhang Jing; Sun Junling; Wu Joseph T; Chang Zhaorui; Liu Fengfeng; Fang Vicky J; Zheng Yingdong; Cowling Benjamin J; Varma Jay K; Farrar Jeremy J; Leung Gabriel M; Yu Hongjie

The Lancet. Infectious diseases (2014), 14 (4), 308-318 ISSN:.

BACKGROUND: Hand, foot, and mouth disease is a common childhood illness caused by enteroviruses. Increasingly, the disease has a substantial burden throughout east and southeast Asia. To better inform vaccine and other interventions, we characterised the epidemiology of hand, foot, and mouth disease in China on the basis of enhanced surveillance. METHODS: We extracted epidemiological, clinical, and laboratory data from cases of hand, foot, and mouth disease reported to the Chinese Center for Disease Control and Prevention between Jan 1, 2008, and Dec 31, 2012. We then compiled climatic, geographical, and demographic information. All analyses were stratified by age, disease severity, laboratory confirmation status, and enterovirus serotype. FINDINGS: The surveillance registry included 7,200,092 probable cases of hand, foot, and mouth disease (annual incidence, 1·2 per 1000 person-years from 2010-12), of which 267,942 (3·7%) were laboratory confirmed and 2457 (0·03%) were fatal. Incidence and mortality were highest in children aged 12-23 months (38·2 cases per 1000 person-years and 1·5 deaths per 100,000 person-years in 2012). Median duration from onset to diagnosis was 1·5 days (IQR 0·5-2·5) and median duration from onset to death was 3·5 days (2·5-4·5). The absolute number of patients with cardiopulmonary or neurological complications was 82,486 (case-severity rate 1·1%), and 2457 of 82486 patients with severe disease died (fatality rate 3·0%); 1617 of 1737 laboratory confirmed deaths (93%) were associated with enterovirus 71. Every year in June, hand, foot, and mouth disease peaked in north China, whereas southern China had semiannual outbreaks in May and September-October. Geographical differences in seasonal patterns were weakly associated with climate and demographic factors (variance explained 8-23% and 3-19%, respectively). INTERPRETATION: This is the largest population-based study up to now of the epidemiology of hand, foot, and mouth disease. Future mitigation policies should take into account the heterogeneities of disease burden identified. Additional epidemiological and serological studies are warranted to elucidate the dynamics and immunity patterns of local hand, foot, and mouth disease and to optimise interventions. FUNDING: China-US Collaborative Program on Emerging and Re-emerging Infectious Diseases, WHO, The Li Ka Shing Oxford Global Health Programme and Wellcome Trust, Harvard Center for Communicable Disease Dynamics, and Health and Medical Research Fund, Government of Hong Kong Special Administrative Region.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2cvgvFylsQ%253D%253D&md5=322cba95a890a6e8aac964d3030a8764
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Massilamany, C. ; Gangaplara, A. ; Reddy, J. Intricacies of cardiac damage in coxsackievirus B3 infection: implications for therapy. Int. J. Cardiol. 2014, 177 , 330– 339, DOI: 10.1016/j.ijcard.2014.09.136

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Intricacies of cardiac damage in coxsackievirus B3 infection: implications for therapy

Massilamany Chandirasegaran; Reddy Jay; Gangaplara Arunakumar

International journal of cardiology (2014), 177 (2), 330-339 ISSN:.

Heart disease is the leading cause of death in humans, and myocarditis is one predominant cause of heart failure in young adults. Patients affected with myocarditis can develop dilated cardiomyopathy (DCM), a common reason for heart transplantation, which to date is the only viable option for combatting DCM. Myocarditis/DCM patients show antibodies to coxsackievirus B (CVB)3 and cardiac antigens, suggesting a role for CVB-mediated autoimmunity in the disease pathogenesis; however, a direct causal link remains to be determined clinically. Experimentally, myocarditis can be induced in susceptible strains of mice using the human isolates of CVB3, and the disease pathogenesis of postinfectious myocarditis resembles that of human disease, making the observations made in animals relevant to humans. In this review, we discuss the complex nature of CVB3-induced myocarditis as it relates to the damage caused by both the virus and the host's response to infection. Based on recent data we obtained in the mouse model of CVB3 infection, we provide evidence to suggest that CVB3 infection accompanies the generation of cardiac myosin-specific CD4 T cells that can transfer the disease to naive recipients. The therapeutic implications of these observations are also discussed.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MvnslGgsg%253D%253D&md5=16e7145787e7ae4cee461cead70151f6
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Coleman, L. ; Laing, I. A. ; Bosco, A. Rhinovirus-induced asthma exacerbations and risk populations. Curr. Opin. Allergy Clin. Immunol. 2016, 16 , 179– 185, DOI: 10.1097/ACI.0000000000000245

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17

Rhinovirus-induced asthma exacerbations and risk populations

Coleman, Laura; Laing, Ingrid A.; Bosco, Anthony

Current Opinion in Allergy and Clinical Immunology (2016), 16 (2), v179-185CODEN: COACCS; ISSN:1473-6322. (Lippincott Williams & Wilkins)

Purpose of review: This article discusses recent findings into the mechanisms that det. how viruses trigger asthma exacerbations. Recent findings: Substantial progress has been made in our understanding of the pathogenesis of virus-induced asthma exacerbations. This includes new insights into the role of bacteria, the regulation of interferon responses, and the discovery of innate immune pathways that link viral infections with allergic inflammation. Progress has also been made in elucidating the genetic risk factors for asthma exacerbations, most notably the contribution of the ORMDL3/GSDMB locus on 17q, the mechanisms underlying the farming effect, and the discovery that CDHR3 binds to rhinovirus species C. Summary: Asthma exacerbations are heterogeneous conditions that involve the complex interplay between environmental exposures and innate and adaptive immune function in genetically predisposed individuals. Recent insights into the interrelationships between these factors provide new opportunities for therapeutic intervention.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjsVGitrs%253D&md5=befc250375d80421b652b039536455d8
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Greninger, A. L. ; Naccache, S. N. ; Messacar, K. ; Clayton, A. ; Yu, G. ; Somasekar, S. ; Federman, S. ; Stryke, D. ; Anderson, C. ; Yagi, S. ; Messenger, S. ; Wadford, D. ; Xia, D. ; Watt, J. P. ; Van Haren, K. ; Dominguez, S. R. ; Glaser, C. ; Aldrovandi, G. ; Chiu, C. Y. A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012–14): a retrospective cohort study. Lancet Infect. Dis. 2015, 15 , 671– 682, DOI: 10.1016/S1473-3099(15)70093-9

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A novel outbreak enterovirus D68 strain associated with acute flaccid myelitis cases in the USA (2012-14): a retrospective cohort study

Greninger Alexander L; Naccache Samia N; Yu Guixia; Somasekar Sneha; Federman Scot; Stryke Doug; Messacar Kevin; Dominguez Samuel R; Clayton Anna; Anderson Christopher; Yagi Shigeo; Messenger Sharon; Wadford Debra; Xia Dongxiang; Watt James P; Glaser Carol; Van Haren Keith; Aldrovandi Grace; Chiu Charles Y

The Lancet. Infectious diseases (2015), 15 (6), 671-82 ISSN:.

BACKGROUND: Enterovirus D68 was implicated in a widespread outbreak of severe respiratory illness across the USA in 2014 and has also been reported sporadically in patients with acute flaccid myelitis. We aimed to investigate the association between enterovirus D68 infection and acute flaccid myelitis during the 2014 enterovirus D68 respiratory outbreak in the USA. METHODS: Patients with acute flaccid myelitis who presented to two hospitals in Colorado and California, USA, between Nov 24, 2013, and Oct 11, 2014, were included in the study. Additional cases identified from Jan 1, 2012, to Oct 4, 2014, via statewide surveillance were provided by the California Department of Public Health. We investigated the cause of these cases by metagenomic next-generation sequencing, viral genome recovery, and enterovirus D68 phylogenetic analysis. We compared patients with acute flaccid myelitis who were positive for enterovirus D68 with those with acute flaccid myelitis but negative for enterovirus D68 using the two-tailed Fisher's exact test, two-sample unpaired t test, and Mann-Whitney U test. FINDINGS: 48 patients were included: 25 with acute flaccid myelitis, two with enterovirus-associated encephalitis, five with enterovirus-D68-associated upper respiratory illness, and 16 with aseptic meningitis or encephalitis who tested positive for enterovirus. Enterovirus D68 was detected in respiratory secretions from seven (64%) of 11 patients comprising two temporally and geographically linked acute flaccid myelitis clusters at the height of the 2014 outbreak, and from 12 (48%) of 25 patients with acute flaccid myelitis overall. Phylogenetic analysis revealed that all enterovirus D68 sequences associated with acute flaccid myelitis grouped into a clade B1 strain that emerged in 2010. Of six coding polymorphisms in the clade B1 enterovirus D68 polyprotein, five were present in neuropathogenic poliovirus or enterovirus D70, or both. One child with acute flaccid myelitis and a sibling with only upper respiratory illness were both infected by identical enterovirus D68 strains. Enterovirus D68 viraemia was identified in a child experiencing acute neurological progression of his paralytic illness. Deep metagenomic sequencing of cerebrospinal fluid from 14 patients with acute flaccid myelitis did not reveal evidence of an alternative infectious cause to enterovirus D68. INTERPRETATION: These findings strengthen the putative association between enterovirus D68 and acute flaccid myelitis and the contention that acute flaccid myelitis is a rare yet severe clinical manifestation of enterovirus D68 infection in susceptible hosts. FUNDING: National Institutes of Health, University of California, Abbott Laboratories, and the Centers for Disease Control and Prevention.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2MjgsFKhtQ%253D%253D&md5=83bb81d7b5f6db5bb1c80896cb4ce2b1
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Chang, L. Y. ; Lin, T. Y. ; Hsu, K. H. ; Huang, Y. C. ; Lin, K. L. ; Hsueh, C. ; Shih, S. R. ; Ning, H. C. ; Hwang, M. S. ; Wang, H. S. ; Lee, C. Y. Clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease. Lancet 1999, 354 , 1682– 1686, DOI: 10.1016/S0140-6736(99)04434-7

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Clinical features and risk factors of pulmonary oedema after enterovirus-71-related hand, foot, and mouth disease

Chang L Y; Lin T Y; Hsu K H; Huang Y C; Lin K L; Hsueh C; Shih S R; Ning H C; Hwang M S; Wang H S; Lee C Y

Lancet (London, England) (1999), 354 (9191), 1682-6 ISSN:0140-6736.

BACKGROUND: In Taiwan, from April to July, 1998, an epidemic of hand, foot, and mouth disease associated with enterovirus 71 (EV71) occurred with fatal complications. We did a clinical study of EV71-related diseases in Taiwan. METHODS: We studied 154 children with virus-culture confirmed EV71 infection. Children were divided into three groups: 11 patients with pulmonary oedema; 38 patients with central nervous system (CNS) involvement and no pulmonary oedema; and 105 children without complications. We compared the clinical features, laboratory findings, risk factors, and outcome among these three groups. FINDINGS: Nine children with pulmonary oedema had hand, foot, and mouth disease, one had herpangina, and one had febrile illness with eight children with limb weakness and one with limb hypesthesia. All children had had sudden onset of tachycardia, tachypnoea, and cyanosis 1-3 days after onset of the disease. Nine of 11 children died within 12 h of intubation; one child was braindead within 15 h and died 17 days after intubation; one child was in deep coma and died 3 months later. In children with CNS complication and no pulmonary oedema, one child died of pneumonia after 4 months of ventilator support and four children had sequelae. All 105 children without complications recovered. There was a significant association between CNS involvement and pulmonary oedema (odds ratio 12.4 [95% CI 2.6-60.1], p=0.001). Risk factors for pulmonary oedema after CNS involvement were hyperglycaemia, leucocytosis, and limb weakness. Hyperglycaemia was the most significant prognostic factor for pulmonary oedema (odds ratio 21.5 [3-159], p=0.003). INTERPRETATION: EV71 can cause hand, foot, and mouth disease, CNS involvement with severe sequelae, and fatal pulmonary oedema. Hyperglycaemia is the most important prognostic factor.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD3c%252Fjtlaiug%253D%253D&md5=3e90d1d63f5e614cb59e2fb5b325cdb9
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Wong, K. T. ; Munisamy, B. ; Ong, K. C. ; Kojima, H. ; Noriyo, N. ; Chua, K. B. ; Ong, B. B. ; Nagashima, K. The distribution of inflammation and virus in human enterovirus 71 encephalomyelitis suggests possible viral spread by neural pathways. J. Neuropathol. Exp. Neurol. 2008, 67 , 162– 169, DOI: 10.1097/nen.0b013e318163a990

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The distribution of inflammation and virus in human enterovirus 71 encephalomyelitis suggests possible viral spread by neural pathways

Wong Kum Thong; Munisamy Badmanathan; Ong Kien Chai; Kojima Hideaki; Noriyo Nagata; Chua Kaw Bing; Ong Beng Beng; Nagashima Kazuo

Journal of neuropathology and experimental neurology (2008), 67 (2), 162-9 ISSN:0022-3069.

Previous neuropathologic studies of Enterovirus 71 encephalomyelitis have not investigated the anatomic distribution of inflammation and viral localization in the central nervous system (CNS) in detail. We analyzed CNS and non-CNS tissues from 7 autopsy cases from Malaysia and found CNS inflammation patterns to be distinct and stereotyped. Inflammation was most marked in spinal cord gray matter, brainstem, hypothalamus, and subthalamic and dentate nuclei; it was focal in the cerebrum, mainly in the motor cortex, and was rare in dorsal root ganglia. Inflammation was absent in the cerebellar cortex, thalamus, basal ganglia, peripheral nerves, and autonomic ganglia. The parenchymal inflammatory response consisted of perivascular cuffs, variable edema, neuronophagia, and microglial nodules. Inflammatory cells were predominantly CD68-positive macrophage/microglia, but there were a few CD8-positive lymphocytes. There were no viral inclusions; viral antigens and RNA were localized only in the somata and processes of small numbers of neurons and in phagocytic cells. There was no evidence of virus in other CNS cells, peripheral nerves, dorsal root autonomic ganglia, or non-CNS organs. The results indicate that Enterovirus 71 is neuronotropic, and that, although hematogenous spread cannot be excluded, viral spread into the CNS could be via neural pathways, likely the motor but not peripheral sensory or autonomic pathways. Viral spread within the CNS seems to involve motor and possibly other pathways.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1c7ovFehsw%253D%253D&md5=c3417074d44a5ab8b00606780816166a
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Gorbalenya, A. E. ; Donchenko, A. P. ; Blinov, V. M. ; Koonin, E. V. Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold. FEBS Lett. 1989, 243 , 103– 114, DOI: 10.1016/0014-5793(89)80109-7

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Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold

Gorbalenya, A. E.; Donchenko, A. P.; Blinov, V. M.; Kunin, E. V.

FEBS Letters (1989), 243 (2), 103-14CODEN: FEBLAL; ISSN:0014-5793.

Evidence is presented, based on sequence comparison and secondary structure prediction, of a structural and evolutionary relation between chymotrypsin-like serine proteases, cysteine proteases of pos.-strand RNA viruses (3C proteases of picornaviruses and related enzymes of como-, nepo- and potyviruses), and putative serine protease of a sobemovirus. These observations lead to reidentification of the principal catalytic residues of viral proteases. Instead of the pair cysteine and histidine, both located in the C-terminal part of 3C proteases, a triad of conserved histidine, aspartate(glutamate), and cysteine (serine) residues has been identified, the 1st 2 residues resident in the N-terminus, and cysteine in the C-terminal β-barrel domain. These residues are suggested to form a charge-transfer system similar to that formed by the catalytic triad of chymotrypsin-like proteases. Based on the structural analogy with chymotrypsin-like proteases, the histidine residue previously implicated in catalysis, together with 2 partially conserved glycine residues, is predicted to constitute part of the substrate-binding pocket of 3C proteases. A partially conserved threonine-lysine/arginine dipeptide located in the loop preceding the catalytic cysteine is suggested to confer the primary cleavage specificity of 3C toward glutamine/glycine (serine) sites. These observations provide the 1st example of relatedness between proteases belonging, by definition, to different classes.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXit1Olurg%253D&md5=50ed142b77661f7c6a43b12befb603d9
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Anand, K. ; Palm, G. J. ; Mesters, J. R. ; Siddell, S. G. ; Ziebuhr, J. ; Hilgenfeld, R. Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain. EMBO J. 2002, 21 , 3213– 3224, DOI: 10.1093/emboj/cdf327

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Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra α-helical domain

Anand, Kanchan; Palm, Gottfried J.; Mesters, Jeroen R.; Siddell, Stuart G.; Ziebuhr, John; Hilgenfeld, Rolf

EMBO Journal (2002), 21 (13), 3213-3224CODEN: EMJODG; ISSN:0261-4189. (Oxford University Press)

The key enzyme in coronavirus polyprotein processing is the viral main proteinase, Mpro, a protein with extremely low sequence similarity to other viral and cellular proteinases. Here, the crystal structure of the 33.1 kDa transmissible gastroenteritis (corona)virus Mpro is reported. The structure was refined to 1.96 Å resoln. and revealed three dimers in the asym. unit. The mutual arrangement of the protomers in each of the dimers suggests that Mpro self-processing occurs in trans. The active site, comprised of Cys144 and His41, is part of a chymotrypsin-like fold that is connected by a 16 residue loop to an extra domain featuring a novel α-helical fold. Mol. modeling and mutagenesis data implicate the loop in substrate binding and elucidate S1 and S2 subsites suitable to accommodate the side chains of the P1 glutamine and P2 leucine residues of Mpro substrates. Interactions involving the N-terminus and the α-helical domain stabilize the loop in the orientation required for trans-cleavage activity. The study illustrates that RNA viruses have evolved unprecedented variations of the classical chymotrypsin fold.

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Hayden, F. G. ; Turner, R. B. ; Gwaltney, J. M. ; Chi-Burris, K. ; Gersten, M. ; Hsyu, P. ; Patick, A. K. ; Smith, G. J. ; Zalman, L. S. Phase II, randomized, double-blind, placebo-controlled studies of rupintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers. Antimicrob. Agents Chemother. 2003, 47 , 3907– 3916, DOI: 10.1128/AAC.47.12.3907-3916.2003

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Phase II, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2% suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers

Hayden, Frederick G.; Turner, Ronald B.; Gwaltney, Jack M.; Chi-burris, Kathy; Gersten, Merril; Hsyu, Poe; Patick, Amy K.; Smith, George J., III; Zalman, Leora S.

Antimicrobial Agents and Chemotherapy (2003), 47 (12), 3907-3916CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)

Human rhinovirus (HRV) infections are usually self-limited but may be assocd. with serious consequences, particularly in those with asthma and chronic respiratory disease. Effective antiviral agents are needed for preventing and treating HRV illnesses. Ruprintrivir (Agouron Pharmaceuticals, Inc., San Diego, Calif.) selectively inhibits HRV 3C protease and shows potent, broad-spectrum anti-HRV activity in vitro. We conducted three double-blind, placebo-controlled clin. trials in 202 healthy volunteers to assess the activity of ruprintrivir in exptl. HRV infection. Subjects were randomized to receive intranasal ruprintrivir (8 mg) or placebo sprays as prophylaxis (two or five times daily [2×/day or 5×/day] for 5 days) starting 6 h before infection or as treatment (5×/day for 4 days) starting 24 h after infection. Ruprintrivir prophylaxis reduced the proportion of subjects with pos. viral cultures (for 5×/day dosing groups, 44% for ruprintrivir treatment group vs. 70% for placebo treatment group [P = 0.03]; for 2×/day dosing groups, 60% for ruprintrivir group vs. 92% for placebo group [P = 0.004]) and viral titers but did not decrease the frequency of colds. Ruprintrivir treatment reduced the mean total daily symptom score (2.2 for ruprintrivir treatment group and 3.3 for the placebo treatment group [P = 0.014]) by 33%. Secondary endpoints, including viral titers, individual symptom scores, and nasal discharge wts., were also reduced by ruprintrivir treatment. Overall, ruprintrivir was well tolerated; blood-tinged mucus and nasal passage irritation were the most common adverse effects reported. Pharmacokinetic anal. of plasma and nasal ruprintrivir concns. revealed intranasal drug residence with minimal systemic absorption. Results from these studies in exptl. rhinoviral infection support continued investigation of intranasal ruprintrivir in the setting of natural HRV infection.

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Kim, Y. ; Liu, H. ; Galasiti Kankanamalage, A. C. ; Weerasekara, S. ; Hua, D. H. ; Groutas, W. C. ; Chang, K. O. ; Pedersen, N. C. Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor. PLoS Pathog. 2016, 12 , e1005531 DOI: 10.1371/journal.ppat.1005531

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Reversal of the progression of fatal coronavirus infection in cats by a broad-spectrum coronavirus protease inhibitor

Kim, Yunjeong; Liu, Hongwei; Kankanamalage, Anushka C. Galasiti; Weerasekara, Sahani; Hua, Duy H.; Groutas, William C.; Chang, Kyeong-Ok; Pedersen, Niels C.

PLoS Pathogens (2016), 12 (3), e1005531/1-e1005531/18CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)

Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately assocd. with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in lab. cats with FIP. Exptl. FIP is 100% fatal once certain clin. and lab. signs become apparent.We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was assocd. with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant redn. in viral titers was also obsd. in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compds. for further development for important coronaviruses in animals and humans.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtlGru7fN&md5=7900b2718caeaf528f10f553e0e8fa16
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Yang, H. ; Yang, M. ; Ding, Y. ; Liu, Y. ; Lou, Z. ; Zhou, Z. ; Sun, L. ; Mo, L. ; Ye, S. ; Pang, H. ; Gao, G. F. ; Anand, K. ; Bartlam, M. ; Hilgenfeld, R. ; Rao, Z. The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 , 13190– 13195, DOI: 10.1073/pnas.1835675100

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The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor

Yang, Haitao; Yang, Maojun; Ding, Yi; Liu, Yiwei; Lou, Zhiyong; Zhou, Zhe; Sun, Lei; Mo, Lijuan; Ye, Sheng; Pang, Hai; Gao, George F.; Anand, Kanchan; Bartlam, Mark; Hilgenfeld, Rolf; Rao, Zihe

Proceedings of the National Academy of Sciences of the United States of America (2003), 100 (23), 13190-13195CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)

A newly identified severe acute respiratory syndrome coronavirus (SARS-CoV), is the etiol. agent responsible for the outbreak of SARS. The SARS-CoV main protease, which is a 33.8-kDa protease (also called the 3C-like protease), plays a pivotal role in mediating viral replication and transcription functions through extensive proteolytic processing of two replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Here, the authors report the crystal structures of the SARS-CoV main protease at different pH values and in complex with a specific inhibitor. The protease structure has a fold that can be described as an augmented serine-protease, but with a Cys-His at the active site. This series of crystal structures, which is the first, to the authors' knowledge, of any protein from the SARS virus, reveal substantial pH-dependent conformational changes, and an unexpected mode of inhibitor binding, providing a structural basis for rational drug design.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXptFOju7s%253D&md5=b9eb74d9a519f31d3eeca5e26bc7d570
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Tan, J. ; Verschueren, K. H. ; Anand, K. ; Shen, J. ; Yang, M. ; Xu, Y. ; Rao, Z. ; Bigalke, J. ; Heisen, B. ; Mesters, J. R. ; Chen, K. ; Shen, X. ; Jiang, H. ; Hilgenfeld, R. pH-dependent conformational flexibility of the SARS-CoV main proteinase M^pro dimer: molecular dynamics simulations and multiple X-ray structure analyses. J. Mol. Biol. 2005, 354 , 25– 40, DOI: 10.1016/j.jmb.2005.09.012

[Crossref], [PubMed], [CAS], Google Scholar

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pH-dependent Conformational Flexibility of the SARS-CoV Main Proteinase (Mpro) Dimer: Molecular Dynamics Simulations and Multiple X-ray Structure Analyses

Tan, Jinzhi; Verschueren, Koen H. G.; Anand, Kanchan; Shen, Jianhua; Yang, Maojun; Xu, Yechun; Rao, Zihe; Bigalke, Janna; Heisen, Burkhard; Mesters, Jeroen R.; Chen, Kaixian; Shen, Xu; Jiang, Hualiang; Hilgenfeld, Rolf

Journal of Molecular Biology (2005), 354 (1), 25-40CODEN: JMOBAK; ISSN:0022-2836. (Elsevier B.V.)

The SARS coronavirus main proteinase (Mpro) is a key enzyme in the processing of the viral polyproteins and thus an attractive target for the discovery of drugs directed against SARS. The enzyme has been shown by x-ray crystallog. to undergo significant pH-dependent conformational changes. Here, we assess the conformational flexibility of the Mpro by anal. of multiple crystal structures (including two new crystal forms) and by mol. dynamics (MD) calcns. The MD simulations take into account the different protonation states of two histidine residues in the substrate-binding site and explain the pH-activity profile of the enzyme. The low enzymic activity of the Mpro monomer and the need for dimerization are also discussed.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhtFKmsL7I&md5=38db21c716511e9322d97efb85c52a0e
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Hilgenfeld, R. From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design. FEBS J. 2014, 281 , 4085– 4096, DOI: 10.1111/febs.12936

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From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design

Hilgenfeld, Rolf

FEBS Journal (2014), 281 (18), 4085-4096CODEN: FJEOAC; ISSN:1742-464X. (Wiley-Blackwell)

A review. Here, the author focuses on the important contributions that macromol. crystallog. has made over the past 12 yr to elucidating structures and mechanisms of the essential proteases of coronaviruses, the main protease (Mpro) and the papain-like protease (PLpro). The role of x-ray crystallog. in structure-assisted drug discovery against these targets is discussed. Aspects dealt with in this review include the emergence of the SARS coronavirus in 2002-2003 and of the MERS coronavirus 10 yr later and the origins of these viruses. The crystal structure of the free SARS coronavirus Mpro and its dependence on pH is discussed, as are efforts to design inhibitors on the basis of these structures. The mechanism of maturation of the enzyme from the viral polyprotein is still a matter of debate. The crystal structure of the SARS coronavirus PLpro and its complex with ubiquitin is also discussed, as is its ortholog from MERS coronavirus. Efforts at predictive structure-based inhibitor development for bat coronavirus Mpros to increase the preparedness against zoonotic transmission to man are described as well. The paper closes with a brief discussion of structure-based discovery of antivirals in an academic setting.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFKnsbbI&md5=6a9330976a5c3a13dd4bf7bed14665ce
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Anand, K. ; Ziebuhr, J. ; Wadhwani, P. ; Mesters, J. R. ; Hilgenfeld, R. Coronavirus main proteinase (3CL^pro) structure: basis for design of anti-SARS drugs. Science 2003, 300 , 1763– 1767, DOI: 10.1126/science.1085658

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Coronavirus Main Proteinase (3CLpro) Structure: Basis for Design of Anti-SARS Drugs

Anand, Kanchan; Ziebuhr, John; Wadhwani, Parvesh; Mesters, Jeroen R.; Hilgenfeld, Rolf

Science (Washington, DC, United States) (2003), 300 (5626), 1763-1767CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)

A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. The authors detd. crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and the authors constructed a homol. model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Mol. modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXksVKisLk%253D&md5=7a8d8d39a783cb3960f6f22931f35569
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Tan, J. ; George, S. ; Kusov, Y. ; Perbandt, M. ; Anemuller, S. ; Mesters, J. R. ; Norder, H. ; Coutard, B. ; Lacroix, C. ; Leyssen, P. ; Neyts, J. ; Hilgenfeld, R. 3C protease of enterovirus 68: structure-based design of Michael acceptor inhibitors and their broad-spectrum antiviral effects against picornaviruses. J. Virol. 2013, 87 , 4339– 4351, DOI: 10.1128/JVI.01123-12

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3C protease of enterovirus 68: structure-based design of Michael acceptor inhibitors and their broad-spectrum antiviral effects against picornaviruses

Tan, Jinzhi; George, Shyla; Kusov, Yuri; Perbandt, Markus; Anemueller, Stefan; Mesters, Jeroen R.; Norder, Helene; Coutard, Bruno; Lacroix, Celine; Leyssen, Pieter; Neyts, Johan; Hilgenfeld, Rolf

Journal of Virology (2013), 87 (8), 4339-4351CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)

The authors detd. the cleavage specificity and the crystal structure of the 3C protease of enterovirus 68 (EV68 3Cpro). The protease exhibits a typical chymotrypsin fold with a Cys...His...Glu catalytic triad; its three-dimensional structure is closely related to that of the 3Cpro of rhinovirus 2, as well as to that of poliovirus. The phylogenetic position of the EV68 3Cpro between the corresponding enzymes of rhinoviruses on the one hand and classical enteroviruses on the other prompted the authors to use the crystal structure for the design of irreversible inhibitors, with the goal of discovering broad-spectrum antiviral compds. The authors synthesized a series of peptidic α,β-unsatd. Et esters of increasing length and for each inhibitor candidate, the authors detd. a crystal structure of its complex with the EV68 3Cpro, which served as the basis for the next design round. To exhibit inhibitory activity, compds. must span at least P3 to P1'; the most potent inhibitors comprise P4 to P1'. Inhibitory activities were found against the purified 3C protease of EV68, as well as with replicons for poliovirus and EV71 (50% effective concn. [EC50] = 0.5 μM for the best compd.). Antiviral activities were detd. using cell cultures infected with EV71, poliovirus, echovirus 11, and various rhinovirus serotypes. The most potent inhibitor, SG85, exhibited activity with EC50s of ≈180 nM against EV71 and ≈60 nM against human rhinovirus 14 in a live virus-cell-based assay. Even the shorter SG75, spanning only P3 to P1', displayed significant activity (EC50 = 2 to 5 μM) against various rhinoviruses.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotVyqtr4%253D&md5=74bfd16bb98e1bd5b12051f6edc6fbe9
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Lu, G. ; Qi, J. ; Chen, Z. ; Xu, X. ; Gao, F. ; Lin, D. ; Qian, W. ; Liu, H. ; Jiang, H. ; Yan, J. ; Gao, G. F. Enterovirus 71 and Coxsackievirus A16 3C proteases: binding to rupintrivir and their substrates and anti-hand, foot, and mouth disease virus drug design. J. Virol. 2011, 85 , 10319– 10331, DOI: 10.1128/JVI.00787-11

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Enterovirus 71 and coxsackievirus A16 3C proteases: binding to rupintrivir and their substrates and anti-hand, foot, and mouth disease virus drug design

Lu, Guangwen; Qi, Jianxun; Chen, Zhujun; Xu, Xiang; Gao, Feng; Lin, Daizong; Qian, Wangke; Liu, Hong; Jiang, Hualiang; Yan, Jinghua; Gao, George F.

Journal of Virology (2011), 85 (19), 10319-10331CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the major causative agents of hand, foot, and mouth disease (HFMD), which is prevalent in Asia. Thus far, there are no prophylactic or therapeutic measures against HFMD. The 3C proteases from EV71 and CVA16 play important roles in viral replication and are therefore ideal drug targets. By using biochem., mutational, and structural approaches, we broadly characterized both proteases. A series of high-resoln. structures of the free or substrate-bound enzymes were solved. These structures, together with our cleavage specificity assay, well explain the marked substrate preferences of both proteases for particular P4, P1, and P1' residue types, as well as the relative malleability of the P2 amino acid. More importantly, the complex structures of EV71 and CVA16 3Cs with rupintrivir, a specific human rhinovirus (HRV) 3C protease inhibitor, were solved. These structures reveal a half-closed S2 subsite and a size-reduced S1' subsite that limit the access of the P1' group of rupintrivir to both enzymes, explaining the reported low inhibition activity of the compd. toward EV71 and CVA16. In conclusion, the detailed characterization of both proteases in this study could direct us to a proposal for rational design of EV71/CVA16 3C inhibitors.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlWiurvI&md5=9e9cee092cfbc470f57ecaddd32064e4
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Wang, F. ; Chen, C. ; Tan, W. ; Yang, K. ; Yang, H. Structure of main protease from human coronavirus NL63: insights for wide spectrum anti-coronavirus drug design. Sci. Rep. 2016, 6 , 22677, DOI: 10.1038/srep22677

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Structure of Main Protease from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Drug Design

Wang, Fenghua; Chen, Cheng; Tan, Wenjie; Yang, Kailin; Yang, Haitao

Scientific Reports (2016), 6 (), 22677CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)

First identified in The Netherlands in 2004, human coronavirus NL63 (HCoV-NL63) was found to cause worldwide infections. Patients infected by HCoV-NL63 are typically young children with upper and lower respiratory tract infection, presenting with symptoms including croup, bronchiolitis, and pneumonia. Unfortunately, there are currently no effective antiviral therapy to contain HCoV-NL63 infection. CoV genomes encode an integral viral component, main protease (Mpro), which is essential for viral replication through proteolytic processing of RNA replicase machinery. Due to the sequence and structural conservation among all CoVs, Mpro has been recognized as an attractive mol. target for rational anti-CoV drug design. Here we present the crystal structure of HCoV-NL63 Mpro in complex with a Michael acceptor inhibitor N3. Structural anal., consistent with biochem. inhibition results, reveals the mol. mechanism of enzyme inhibition at the highly conservative substrate-recognition pocket. We show such mol. target remains unchanged across 30 clin. isolates of HCoV-NL63 strains. Through comparative study with Mpros from other human CoVs (including the deadly SARS-CoV and MERS-CoV) and their related zoonotic CoVs, our structure of HCoV-NL63 Mpro provides crit. insight into rational development of wide spectrum antiviral therapeutics to treat infections caused by human CoVs.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjslGrtLg%253D&md5=b756a48075e0f5551871a9569a99c8c0
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Dragovich, P. S. ; Zhou, R. ; Skalitzky, D. J. ; Fuhrman, S. A. ; Patick, A. K. ; Ford, C. E. ; Meador, J. W., 3rd ; Worland, S. T. Solid-phase synthesis of irreversible human rhinovirus 3C protease inhibitors. Part 1: Optimization of tripeptides incorporating N-terminal amides. Bioorg. Med. Chem. 1999, 7 , 589– 598, DOI: 10.1016/S0968-0896(99)00005-X

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Solid-phase synthesis of irreversible human rhinovirus 3C protease inhibitors. Part 1: Optimization of tripeptides incorporating N-terminal amides

Dragovich, Peter S.; Zhou, Ru; Skalitzky, Donald J.; Fuhrman, Shella A.; Patick, Amy K.; Ford, Clifford E.; Meador, James W., III; Worland, Stephen T.

Bioorganic & Medicinal Chemistry (1999), 7 (4), 589-598CODEN: BMECEP; ISSN:0968-0896. (Elsevier Science Ltd.)

The optimization of a series of irreversible human rhinovirus (HRV) 3C protease (3CP) inhibitors is described. These inhibitors are comprised of an L-Leu-L-Phe-L-Gln tripeptide contg. an N-terminal amide moiety and a C-terminal Et propenoate Michael acceptor. Examn. of approx. 500 compds. with varying N-terminal amides utilizing solid-phase synthesis and high-throughput assay techniques is described along with the soln. phase prepn. of several highly active mols. A tripeptide Michael acceptor contg. an N-terminal amide derived from 5-methyl-isoxazole-3-carboxylic acid is shown to exhibit potent, irreversible anti-3CP activity (kobs/[I]=260,000 M-1 S-1; type-14 3CP) and broad-spectrum anti-rhinoviral properties (av. EC50=0.47 μM against four different HRV serotypes).

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXis12mt7s%253D&md5=baccfe799291b59c3c46a53ca12a9303
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Kusov, Y. ; Tan, J. ; Alvarez, E. ; Enjuanes, L. ; Hilgenfeld, R. A G-quadruplex-binding macrodomain within the ″SARS-unique domain″ is essential for the activity of the SARS-coronavirus replication-transcription complex. Virology 2015, 484 , 313– 322, DOI: 10.1016/j.virol.2015.06.016

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A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex

Kusov, Yuri; Tan, Jinzhi; Alvarez, Enrique; Enjuanes, Luis; Hilgenfeld, Rolf

Virology (2015), 484 (), 313-322CODEN: VIRLAX; ISSN:0042-6822. (Elsevier B.V.)

The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examd. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtVygtLzF&md5=a7704b75b035c65eabfb89602ae27704
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van Kuppeveld, F. J. ; Galama, J. M. ; Zoll, J. ; Melchers, W. J. Genetic analysis of a hydrophobic domain of coxsackie B3 virus protein 2B: a moderate degree of hydrophobicity is required for a cis-acting function in viral RNA synthesis. J. Virol. 1995, 69 , 7782– 7790, DOI: 10.1128/JVI.69.12.7782-7790.1995

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Genetic analysis of a hydrophobic domain of coxsackie B3 virus protein 2B: a moderate degree of hydrophobicity is required for a cis-acting function in viral RNA synthesis

van Kuppeveld, Frank J. M.; Galama, Jochem M. D.; Zoll, Jan; Melchers, Willem J. G.

Journal of Virology (1995), 69 (12), 7782-90CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)

Coxsackie B virus protein 2B contains near its C terminus a hydrophobic domain with an amino acid compn. that is characteristic for transmembrane regions. A mol. genetic approach was followed to define the role of this domain in virus reprodn. and to study the structural and hydrophobic requirements of the domain. Nine substitution mutations were introduced in an infectious cDNA clone of coxsackie B3 virus. The effects of the mutations were studied in vitro by transfection of Buffalo green monkey cells with copy RNA transcripts. The results reported here suggest that a crit. degree of hydrophobicity of the domain is essential for virus growth. The mutations S77M, C75M, I64S, and V66S, which caused either a small increase or decrease in mean hydrophobicity, yielded viable viruses. The double mutations S77M/C75M and I64S/V6-6S, which caused a more pronounced increase or decrease in hydrophobicity, were nonviable. Neg. charged residues (mutations A71E, I73E, and A71E/I73E) abolished virus growth. The mutations had no effect on the synthesis and processing of the viral polyprotein. Replication and complementation were studied by using a subgenomic coxsackievirus replicon contg. the luciferase gene in place of the capsid coding region. Anal. of luciferase accumulation demonstrated that the mutations cause primary defects in viral RNA synthesis that cannot be complemented by wild-type protein 2B provided in trans. The hydrophobic domain is predicted by computer anal. to form a multimeric transmembrane helix. The proposed interaction with the membrane and the implications of the mutations on this interaction are discussed.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptlOkurg%253D&md5=dc173c346abfba17a5ac717b993e27f7
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Lanke, K. H. ; van der Schaar, H. M. ; Belov, G. A. ; Feng, Q. ; Duijsings, D. ; Jackson, C. L. ; Ehrenfeld, E. ; van Kuppeveld, F. J. GBF1, a guanine nucleotide exchange factor for Arf, is crucial for Coxsackievirus B3 RNA replication. J. Virol. 2009, 83 , 11940– 11949, DOI: 10.1128/JVI.01244-09

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GBF1, a guanine nucleotide exchange factor for Arf, is crucial for coxsackievirus B3 RNA replication

Lanke, Kjerstin H. W.; van der Schaar, Hilde M.; Belov, George A.; Feng, Qian; Duijsings, Daniel; Jackson, Catherine L.; Ehrenfeld, Ellie; van Kuppeveld, Frank J. M.

Journal of Virology (2009), 83 (22), 11940-11949CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)

The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a crit. role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Addnl., the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a crit. role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFKqtbs%253D&md5=58e3e840431f2ef1b869b408aac5cb38
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Pettersen, E. F. ; Goddard, T. D. ; Huang, C. C. ; Couch, G. S. ; Greenblatt, D. M. ; Meng, E. C. ; Ferrin, T. E. UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25 , 1605– 1612, DOI: 10.1002/jcc.20084

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UCSF Chimera-A visualization system for exploratory research and analysis

Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.; Couch, Gregory S.; Greenblatt, Daniel M.; Meng, Elaine C.; Ferrin, Thomas E.

Journal of Computational Chemistry (2004), 25 (13), 1605-1612CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)

The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale mol. assemblies such as viral coats, and Collab., which allows researchers to share a Chimera session interactively despite being at sep. locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and assocd. structures; ViewDock, for screening docked ligand orientations; Movie, for replaying mol. dynamics trajectories; and Vol. Viewer, for display and anal. of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmvVOhsbs%253D&md5=944b175f440c1ff323705987cf937ee7
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Zhu, L. ; George, S. ; Schmidt, M. F. ; Al-Gharabli, S. I. ; Rademann, J. ; Hilgenfeld, R. Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease. Antiviral Res. 2011, 92 , 204– 212, DOI: 10.1016/j.antiviral.2011.08.001

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Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease

Zhu, Lili; George, Shyla; Schmidt, Marco F.; Al-Gharabli, Samer I.; Rademann, Joerg; Hilgenfeld, Rolf

Antiviral Research (2011), 92 (2), 204-212CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)

SARS coronavirus main protease (SARS-CoV Mpro) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV Mpro. Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV Mpro requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We detd. four crystal structures of SARS-CoV Mpro in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV Mpro, with Ki values in the μM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2 = Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also detd. a crystal structure of SARS-CoV Mpro in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV Mpro, with Ki = 2.24 ± 0.58 μM. These results show that the stringent substrate specificity of the SARS-CoV Mpro with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the obsd. cleavage specificity of the target protease.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtlKkurfL&md5=2a888389fe963d87d07b8a67b9c0febb
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Qian, J. ; Cuerrier, D. ; Davies, P. L. ; Li, Z. ; Powers, J. C. ; Campbell, R. L. Cocrystal structures of primed side-extending alpha-ketoamide inhibitors reveal novel calpain-inhibitor aromatic interactions. J. Med. Chem. 2008, 51 , 5264– 5270, DOI: 10.1021/jm800045t

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Cocrystal Structures of Primed Side-Extending α-Ketoamide Inhibitors Reveal Novel Calpain-Inhibitor Aromatic Interactions

Qian, Jin; Cuerrier, Dominic; Davies, Peter L.; Li, Zhaozhao; Powers, James C.; Campbell, Robert L.

Journal of Medicinal Chemistry (2008), 51 (17), 5264-5270CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)

Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to Ca2+ signaling. When Ca2+ homeostasis is disrupted, calpain overactivation causes unregulated proteolysis, which can contribute to diseases such as postischemic injury and cataract formation. Potent calpain inhibitors exist, but of these many cross-react with other cysteine proteases and will need modification to specifically target calpain. Here, we present crystal structures of rat calpain 1 protease core (μI-II) bound to two α-ketoamide-based calpain inhibitors contg. adenyl and piperazyl primed-side extensions. An unexpected arom.-stacking interaction is obsd. between the primed-side adenine moiety and the Trp298 side chain. This interaction increased the potency of the inhibitor toward μI-II and heterodimeric m-calpain. Moreover, stacking orients the adenine such that it can be used as a scaffold for designing novel primed-side address regions, which could be incorporated into future inhibitors to enhance their calpain specificity.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXpvFSmu7g%253D&md5=b1980a4b84f1a9642d4b3b781115d004
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Romano, K. P. ; Ali, A. ; Aydin, C. ; Soumana, D. ; Ozen, A. ; Deveau, L. M. ; Silver, C. ; Cao, H. ; Newton, A. ; Petropoulos, C. J. ; Huang, W. ; Schiffer, C. A. The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors. PLoS Pathog. 2012, 8 , e1002832, DOI: 10.1371/journal.ppat.1002832

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The molecular basis of drug resistance against hepatitis C virus NS3/4A protease inhibitors

Romano, Keith P.; Ali, Akbar; Aydin, Cihan; Soumana, Djade; Ozen, Aysegul; Deveau, Laura M.; Silver, Casey; Cao, Hong; Newton, Alicia; Petropoulos, Christos J.; Huang, Wei; Schiffer, Celia A.

PLoS Pathogens (2012), 8 (7), e1002832CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)

Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer resistance to nearly all inhibitors in clin. development. Thus, developing the next-generation of drugs that retain activity against a broader spectrum of resistant viral variants requires a comprehensive understanding of the mol. basis of drug resistance. In this study, 16 high-resoln. crystal structures of four representative protease inhibitors - telaprevir, danoprevir, vaniprevir and MK-5172 - in complex with the wild-type protease and three major drug-resistant variants R155K, A156T and D168A, reveal unique mol. underpinnings of resistance to each drug. The drugs exhibit differential susceptibilities to these protease variants in both enzymic and antiviral assays. Telaprevir, danoprevir and vaniprevir interact directly with sites that confer resistance upon mutation, while MK-5172 interacts in a unique conformation with the catalytic triad. This novel mode of MK-5172 binding explains its retained potency against two multi-drug-resistant variants, R155K and D168A. These findings define the mol. basis of HCV N3/4A protease inhibitor resistance and provide potential strategies for designing robust therapies against this rapidly evolving virus.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhtFeisrbK&md5=15ca5b9d370793cd3fa814755ddf99c6
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Venkatraman, S. ; Bogen, S. L. ; Arasappan, A. ; Bennett, F. ; Chen, K. ; Jao, E. ; Liu, Y. T. ; Lovey, R. ; Hendrata, S. ; Huang, Y. ; Pan, W. ; Parekh, T. ; Pinto, P. ; Popov, V. ; Pike, R. ; Ruan, S. ; Santhanam, B. ; Vibulbhan, B. ; Wu, W. ; Yang, W. ; Kong, J. ; Liang, X. ; Wong, J. ; Liu, R. ; Butkiewicz, N. ; Chase, R. ; Hart, A. ; Agrawal, S. ; Ingravallo, P. ; Pichardo, J. ; Kong, R. ; Baroudy, B. ; Malcolm, B. ; Guo, Z. ; Prongay, A. ; Madison, V. ; Broske, L. ; Cui, X. ; Cheng, K. C. ; Hsieh, Y. ; Brisson, J. M. ; Prelusky, D. ; Korfmacher, W. ; White, R. ; Bogdanowich-Knipp, S. ; Pavlovsky, A. ; Bradley, P. ; Saksena, A. K. ; Ganguly, A. ; Piwinski, J. ; Girijavallabhan, V. ; Njoroge, F. G. Discovery of (1R,5S)-N-[3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]- 3-[2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (SCH 503034), a selective, potent, orally bioavailable hepatitis C virus NS3 protease inhibitor: a potential therapeutic agent for the treatment of hepatitis C infection. J. Med. Chem. 2006, 49 , 6074– 6086, DOI: 10.1021/jm060325b

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Discovery of (1R,5S)-N-[3-Amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]- 3-[2(S)-[[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]- 6,6-dimethyl-3-azabicyclo[3.1.0]hexan-2(S)-carboxamide (SCH 503034), a Selective, Potent, Orally Bioavailable Hepatitis C Virus NS3 Protease Inhibitor: A Potential Therapeutic Agent for the Treatment of Hepatitis C Infection

Venkatraman, Srikanth; Bogen, Stephane L.; Arasappan, Ashok; Bennett, Frank; Chen, Kevin; Jao, Edwin; Liu, Yi-Tsung; Lovey, Raymond; Hendrata, Siska; Huang, Yuhua; Pan, Weidong; Parekh, Tejal; Pinto, Patrick; Popov, Veljko; Pike, Russel; Ruan, Sumei; Santhanam, Bama; Vibulbhan, Bancha; Wu, Wanli; Yang, Weiying; Kong, Jianshe; Liang, Xiang; Wong, Jesse; Liu, Rong; Butkiewicz, Nancy; Chase, Robert; Hart, Andrea; Agrawal, Sony; Ingravallo, Paul; Pichardo, John; Kong, Rong; Baroudy, Bahige; Malcolm, Bruce; Guo, Zhuyan; Prongay, Andrew; Madison, Vincent; Broske, Lisa; Cui, Xiaoming; Cheng, Kuo-Chi; Hsieh, Tony Y.; Brisson, Jean-Marc; Prelusky, Danial; Korfmacher, Walter; White, Ronald; Bogdanowich-Knipp, Susan; Pavlovsky, Anastasia; Bradley, Prudence; Saksena, Anil K.; Ganguly, Ashit; Piwinski, John; Girijavallabhan, Viyyoor; Njoroge, F. George

Journal of Medicinal Chemistry (2006), 49 (20), 6074-6086CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)

Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only therapeutic regimens are s.c. interferon-α or polyethylene glycol (PEG)-interferon-α alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only about 40% of the patients showing sustained virol. response. Herein, the SAR leading to the discovery of SCH 503034 (I), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been advanced to clin. trials in human beings for the treatment of hepatitis C viral infections is described. X-ray structure of I complexed with the NS3 protease and biol. data are also discussed.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XptlOlsLg%253D&md5=e48b2e2d3eb19f7f28aaa76f96d03414
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Mandadapu, S. R. ; Weerawarna, P. M. ; Gunnam, M. R. ; Alliston, K. R. ; Lushington, G. H. ; Kim, Y. ; Chang, K. O. ; Groutas, W. C. Potent inhibition of norovirus 3CL protease by peptidyl alpha-ketoamides and alpha-ketoheterocycles. Bioorg. Med. Chem. Lett. 2012, 22 , 4820– 4826, DOI: 10.1016/j.bmcl.2012.05.055

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Potent inhibition of norovirus 3CL protease by peptidyl α-ketoamides and α-ketoheterocycles

Mandadapu, Sivakoteswara Rao; Weerawarna, Pathum M.; Gunnam, Mallikarjuna Reddy; Alliston, Kevin R.; Lushington, Gerald H.; Kim, Yunjeong; Chang, Kyeong-Ok; Groutas, William C.

Bioorganic & Medicinal Chemistry Letters (2012), 22 (14), 4820-4826CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)

A series of structurally-diverse α-ketoamides and α-ketoheterocycles, e.g., Cbz-L-Leu-L-NHCH(CH2R)CO-R1 (R = 2-oxo-3-pyrrolidinyl; R1 = CONHR2, where R2 = i-Pr, cyclopropyl, Bu, EtO2CCH2, cyclohexyl, Bn, t-Bu or R1 = 2-oxazolyl or 2-thiazolyl) was synthesized and subsequently investigated for inhibitory activity against norovirus 3CL protease in vitro, as well as anti-norovirus activity in a cell-based replicon system. The synthesized compds. were found to inhibit norovirus 3CL protease in vitro and to also exhibit potent anti-norovirus activity in a cell-based replicon system.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XoslSrsr8%253D&md5=431756d7bf0ad00793ddd9988d486d11
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Zeng, D. ; Ma, Y. ; Zhang, R. ; Nie, Q. ; Cui, Z. ; Wang, Y. ; Shang, L. ; Yin, Z. Synthesis and structure-activity relationship of alpha-keto amides as enterovirus 71 3C protease inhibitors. Bioorg. Med. Chem. Lett. 2016, 26 , 1762– 1766, DOI: 10.1016/j.bmcl.2016.02.039

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Synthesis and structure-activity relationship of α-keto amides as enterovirus 71 3C protease inhibitors

Zeng, Debin; Ma, Yuying; Zhang, Rui; Nie, Quandeng; Cui, Zhengjie; Wang, Yaxin; Shang, Luqing; Yin, Zheng

Bioorganic & Medicinal Chemistry Letters (2016), 26 (7), 1762-1766CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)

Peptidomimetic α-keto amides I [R = i-Pr, Bu, cyclohexyl, Ph, PhCH2, Me, n-Pr, cyclopropyl, i-Bu, t-Bu, BuCH2, BuCH2CH2, Me(CH2)11, 4-MeC6H4, 4-O2NC6H4, 2-furanylmethyl; R1 = (E)-PhCH:CH, PhCH2O, t-BuO; R2 = H, F; X = bond, CH2] with lactam-substituted side chains and either an L-phenylalanine or L-p-fluorophenylalanine residue were prepd. using Passerini reactions of nonracemic aldehydes with isocyanides and acetic acid followed by hydrolysis and oxidn. and tested as inhibitors of the enterovirus 71 (hand-foot-mouth disease virus) 3C protease (EV71 3C protease). I were tested for their inhibition of EV71 3C protease, their inhibition of EV71 replication, and their toxicity to normal cells. The effect of the structure of I on inhibition of EV71 was detd.; small moieties were preferred at the C-terminal keto amide and inhibitors contg. L-p-fluorophenylalanine residues had better potency than inhibitors contg. L-phenylalanine residues. I [R = i-Pr, n-Pr, cyclopropyl; R1 = (E)-PhCH:CH; R2 = F; X = CH2] exhibited satisfactory activities, with IC50 values for EV71 3C protease of 1.32-1.88 μM and EC50 values on viral replication of 1.08-1.55 μM, and favorable toxicities (CC50 values > 100 μM).

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjt1WltLs%253D&md5=afbaf7dbbe4e7093b42e9d51e6bfcd5c
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Kim, Y. ; Kankanamalage, A. C. ; Damalanka, V. C. ; Weerawarna, P. M. ; Groutas, W. C. ; Chang, K. O. Potent inhibition of enterovirus D68 and human rhinoviruses by dipeptidyl aldehydes and alpha-ketoamides. Antiviral Res. 2016, 125 , 84– 91, DOI: 10.1016/j.antiviral.2015.11.010

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Potent inhibition of enterovirus D68 and human rhinoviruses by dipeptidyl aldehydes and α-ketoamides

Kim, Yunjeong; Kankanamalage, Anushka C. Galasiti; Damalanka, Vishnu C.; Weerawarna, Pathum M.; Groutas, William C.; Chang, Kyeong-Ok

Antiviral Research (2016), 125 (), 84-91CODEN: ARSRDR; ISSN:0166-3542. (Elsevier B.V.)

Enterovirus D68 (EV-D68) is an emerging pathogen responsible for mild to severe respiratory infections that occur mostly in infants, children and teenagers. EV-D68, one of more than 100 non-polio enteroviruses, is acid-labile and biol. similar to human rhinoviruses (HRV) (originally classified as HRV87). However, there is no approved preventive or therapeutic measure against EV-D68, HRV, or other enteroviruses. In this study, we evaluated the antiviral activity of series of dipeptidyl compds. against EV-D68 and HRV strains, and demonstrated that several peptidyl aldehyde and α-ketoamide peptidyl compds. are potent inhibitors of EV-D68 and HRV strains with high in-vitro therapeutic indexes (>1000). One of the α-ketoamide compds. is shown to have favorable pharmacokinetics profiles, including a favorable oral bioavailability in rats. Recent successful development of α-ketoamide protease inhibitors against hepatitis C virus suggests these compds. may have a high potential for further optimization and development against emerging EV-D68, as well as HRV.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXitVSrs7bK&md5=907c89106582ea1c0b23434ca3394f23
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Kim, Y. ; Lovell, S. ; Tiew, K. C. ; Mandadapu, S. R. ; Alliston, K. R. ; Battaile, K. P. ; Groutas, W. C. ; Chang, K. O. Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses. J. Virol. 2012, 86 , 11754– 11762, DOI: 10.1128/JVI.01348-12

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Broad-spectrum antivirals against 3C or 3C-like proteases of picornaviruses, noroviruses, and coronaviruses

Kim, Yunjeong; Lovell, Scott; Tiew, Kok-Chuan; Mandadapu, Sivakoteswara Rao; Alliston, Kevin R.; Battaile, Kevin P.; Groutas, William C.; Chang, Kyeong-Ok

Journal of Virology (2012), 86 (21), 11754-11762CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)

Phylogenetic anal. has demonstrated that some pos.-sense RNA viruses can be classified into the picornavirus-like super-cluster, which includes picornaviruses, caliciviruses, and coronaviruses. These viruses possess 3C or 3C-like proteases (3Cpro or 3CLpro, resp.), which contain a typical chymotrypsin-like fold and a catalytic triad (or dyad) with a Cys residue as a nucleophile. The conserved key sites of 3Cpro or 3CLpro may serve as attractive targets for the design of broad-spectrum antivirals for multiple viruses in the supercluster. We previously reported the structure-based design and synthesis of potent protease inhibitors of Norwalk virus (NV), a member of the Caliciviridae family. We report herein the broad-spectrum antiviral activities of three compds. possessing a common dipeptidyl residue with different warheads, i.e., an aldehyde (GC373), a bisulfite adduct (GC376), and an α-ketoamide (GC375), against viruses that belong to the supercluster. All compds. were highly effective against the majority of tested viruses, with half-maximal inhibitory concns. in the high nanomolar or low micromolar range in enzyme- and/or cell-based assays and with high therapeutic indexes. We also report the high-resoln. X-ray cocrystal structures of NV 3CLpro-, poliovirus 3Cpro-, and transmissible gastroenteritis virus 3CLpro- GC376 inhibitor complexes, which show the compd. covalently bound to a nucleophilic Cys residue in the catalytic site of the corresponding protease. We conclude that these compds. have the potential to be developed as antiviral therapeutics aimed at a single virus or multiple viruses in the picornavirus-like supercluster by targeting 3Cpro or 3CLpro.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XhsFCmur3M&md5=c7fe984b52490199a2c2ef333c22fb98
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Prior, A. M. ; Kim, Y. ; Weerasekara, S. ; Moroze, M. ; Alliston, K. R. ; Uy, R. A. ; Groutas, W. C. ; Chang, K. O. ; Hua, D. H. Design, synthesis, and bioevaluation of viral 3C and 3C-like protease inhibitors. Bioorg. Med. Chem. Lett. 2013, 23 , 6317– 6320, DOI: 10.1016/j.bmcl.2013.09.070

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Design, synthesis, and bioevaluation of viral 3C and 3C-like protease inhibitors

Prior, Allan M.; Kim, Yunjeong; Weerasekara, Sahani; Moroze, Meghan; Alliston, Kevin R.; Uy, Roxanne Adeline Z.; Groutas, William C.; Chang, Kyeong-Ok; Hua, Duy H.

Bioorganic & Medicinal Chemistry Letters (2013), 23 (23), 6317-6320CODEN: BMCLE8; ISSN:0960-894X. (Elsevier B.V.)

A class of tripeptidyl transition state inhibitors contg. a P1 glutamine surrogate, a P2 leucine, and a P3 arylalanines, was found to potently inhibit Norwalk virus replication in enzyme and cell based assays. An array of warheads, including aldehyde, α-ketoamide, bisulfite adduct, and α-hydroxyphosphonate transition state mimic, was also investigated. Tripeptidyls 2 and 6 possess antiviral activities against noroviruses, human rhinovirus, severe acute respiratory syndrome coronavirus, and coronavirus 229E, suggesting a broad range of antiviral activities.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhs1WjurfI&md5=4e71ce4a328d813f0b53ca153296f55b
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Lee, C. C. ; Kuo, C. J. ; Ko, T. P. ; Hsu, M. F. ; Tsui, Y. C. ; Chang, S. C. ; Yang, S. ; Chen, S. J. ; Chen, H. C. ; Hsu, M. C. ; Shih, S. R. ; Liang, P. H. ; Wang, A. H. Structural basis of inhibition specificities of 3C and 3C-like proteases by zinc-coordinating and peptidomimetic compounds. J. Biol. Chem. 2009, 284 , 7646– 7655, DOI: 10.1074/jbc.M807947200

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Structural Basis of Inhibition Specificities of 3C and 3C-like Proteases by Zinc-coordinating and Peptidomimetic Compounds

Lee, Cheng-Chung; Kuo, Chih-Jung; Ko, Tzu-Ping; Hsu, Min-Feng; Tsui, Yao-Chen; Chang, Shih-Cheng; Yang, Syaulan; Chen, Shu-Jen; Chen, Hua-Chien; Hsu, Ming-Chu; Shih, Shin-Ru; Liang, Po-Huang; Wang, Andrew H.-J.

Journal of Biological Chemistry (2009), 284 (12), 7646-7655CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)

Human coxsackievirus (CV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. In picornavirus, a chymotrypsin-like protease (3Cpro) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. A 3C-like protease (3CLpro) also exists in human coronaviruses (CoV) such as 229E and the one causing severe acute respiratory syndrome (SARS). To combat SARS, we previously had developed peptidomimetic and zinc-coordinating inhibitors of 3CLpro. As shown in the present study, some of these compds. were also found to be active against 3Cpro of CV strain B3 (CVB3). Several crystal structures of 3Cpro from CVB3 and 3CLpro from CoV-229E and SARS-CoV in complex with the inhibitors were solved. The zinc-coordinating inhibitor is tetrahedrally coordinated to the His40-Cys147 catalytic dyad of CVB3 3Cpro. The presence of specific binding pockets for the residues of peptidomimetic inhibitors explains the binding specificity. Our results provide a structural basis for inhibitor optimization and development of potential drugs for antiviral therapies.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXjtVSmsLc%253D&md5=af8f06673d60dbd5d8c19743f62ea015
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Binford, S. L. ; Maldonado, F. ; Brothers, M. A. ; Weady, P. T. ; Zalman, L. S. ; Meador, J. W., 3rd ; Matthews, D. A. ; Patick, A. K. Conservation of amino acids in human rhinovirus 3C protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus 3C protease inhibitor. Antimicrob. Agents Chemother. 2005, 49 , 619– 626, DOI: 10.1128/AAC.49.2.619-626.2005

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Conservation of amino acids in human rhinovirus 3C protease correlates with broad-spectrum antiviral activity of rupintrivir, a novel human rhinovirus 3C protease inhibitor

Binford, S. L.; Maldonado, F.; Brothers, M. A.; Weady, P. T.; Zalman, L. S.; Meador, J. W., III; Matthews, D. A.; Patick, A. K.

Antimicrobial Agents and Chemotherapy (2005), 49 (2), 619-626CODEN: AMACCQ; ISSN:0066-4804. (American Society for Microbiology)

The picornavirus 3C protease is required for the majority of proteolytic cleavages that occur during the viral life cycle. Comparisons of published amino acid sequences from 6 human rhinoviruses (HRV) and 20 human enteroviruses (HEV) show considerable variability in the 3C protease-coding region but strict conservation of the catalytic triad residues. Rupintrivir (formerly AG7088) is an irreversible inhibitor of HRV 3C protease with potent in vitro activity against all HRV serotypes (48 of 48), HEV strains (4 of 4), and untyped HRV field isolates (46 of 46) tested. To better understand the relationship between in vitro antiviral activity and 3C protease-rupintrivir binding interactions, the authors performed nucleotide sequence analyses on an addnl. 21 HRV serotypes and 11 HRV clin. isolates. Antiviral activity was also detd. for 23 HRV clin. isolates and four addnl. HEV strains. Sequence comparison of 3C proteases (n = 58) show that 13 and 11 of the 14 amino acids that are involved in side chain interactions with rupintrivir are strictly conserved among HRV and HEV, resp. These sequence analyses are consistent with the comparable in vitro antiviral potencies of rupintrivir against all HRV serotypes, HRV isolates, and HEV strains tested (50% effective concn. range, 3 to 183 nM; n = 125). In summary, the conservation of crit. amino acid residues in 3C protease and the observation of potent, broad-spectrum antipicornavirus activity of rupintrivir highlight the advantages of 3C protease as an antiviral target.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXht12nt7Y%253D&md5=936a058a019545e186f5a6e5b4cec298
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Xue, X. ; Yang, H. ; Shen, W. ; Zhao, Q. ; Li, J. ; Yang, K. ; Chen, C. ; Jin, Y. ; Bartlam, M. ; Rao, Z. Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction. J. Mol. Biol. 2007, 366 , 965– 975, DOI: 10.1016/j.jmb.2006.11.073

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Production of Authentic SARS-CoV Mpro with Enhanced Activity: Application as a Novel Tag-cleavage Endopeptidase for Protein Overproduction

Xue, Xiaoyu; Yang, Haitao; Shen, Wei; Zhao, Qi; Li, Jun; Yang, Kailin; Chen, Cheng; Jin, Yinghua; Bartlam, Mark; Rao, Zihe

Journal of Molecular Biology (2007), 366 (3), 965-975CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)

The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (Mpro) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiol. agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV Mpro was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV Mpro with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV Mpro with addnl. residues at the N or C terminus. Our results show that addnl. residues at the N terminus, but not at the C terminus, of Mpro are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV Mpro and its complex with a Michael acceptor inhibitor were detd. to 1.6 Å and 1.95 Å resoln. resp. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV Mpro could serve as a new tag-cleavage endopeptidase for protein overprodn., and the WT SARS-CoV Mpro is more appropriate for mechanistic characterization and inhibitor design.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXhtlehsr8%253D&md5=1f9a889dca005727859e239ad9eb8d7d
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Verschueren, K. H. ; Pumpor, K. ; Anemuller, S. ; Chen, S. ; Mesters, J. R. ; Hilgenfeld, R. A structural view of the inactivation of the SARS coronavirus main proteinase by benzotriazole esters. Chem. Biol. 2008, 15 , 597– 606, DOI: 10.1016/j.chembiol.2008.04.011

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49

A Structural View of the Inactivation of the SARS Coronavirus Main Proteinase by Benzotriazole Esters

Verschueren, Koen H. G.; Pumpor, Ksenia; Anemueller, Stefan; Chen, Shuai; Mesters, Jeroen R.; Hilgenfeld, Rolf

Chemistry & Biology (Cambridge, MA, United States) (2008), 15 (6), 597-606CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)

Summary: The main proteinase (Mpro) of the severe acute respiratory syndrome (SARS) coronavirus is a principal target for the design of anticoronaviral compds. Benzotriazole esters have been reported as potent nonpeptidic inhibitors of the enzyme, but their exact mechanism of action remains unclear. Here we present crystal structures of SARS-CoV Mpro, the active-site cysteine of which has been acylated by benzotriazole esters that act as suicide inhibitors. In one of the structures, the thioester product has been hydrolyzed and benzoic acid is obsd. to bind to the hydrophobic S2 pocket. This structure also features the enzyme with a shortened N-terminal segment ("amputated N finger"). The results further the understanding of the important role of the N finger for catalysis as well as the design of benzotriazole inhibitors with improved specificity.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnt1alu74%253D&md5=732c7f2eaa8e5a48aa95bf3a0f9836e7
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Krug, M. ; Weiss, M. S. ; Heinemann, U. ; Mueller, U. XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS. J. Appl. Crystallogr. 2012, 45 , 568– 572, DOI: 10.1107/S0021889812011715

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50

XDSAPP: a graphical user interface for the convenient processing of diffraction data using XDS

Krug, Michael; Weiss, Manfred S.; Heinemann, Udo; Mueller, Uwe

Journal of Applied Crystallography (2012), 45 (3), 568-572CODEN: JACGAR; ISSN:0021-8898. (International Union of Crystallography)

XDSAPP is a Tcl/Tk-based graphical user interface for the easy and convenient processing of diffraction data sets using XDS. It provides easy access to all XDS functionalities, automates the data processing and generates graphical plots of various data set statistics provided by XDS. By incorporating addnl. software, further information on certain features of the data set, such as radiation decay during data collection or the presence of pseudo-translational symmetry and/or twinning, can be obtained. Intensity files suitable for CCP4, CNS and SHELX are generated.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnsFGmtLo%253D&md5=8f0b78fdea7ed6cc580de9eb561e8b9e
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Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67 , 282– 292, DOI: 10.1107/S090744491003982X

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51

An introduction to data reduction: Space-group determination, scaling and intensity statistics

Evans, Philip R.

Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 282-292CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)

A review. This paper presents an overview of how to run the CCP4 programs for data redn. (SCALA, POINTLESS and CTRUNCATE) through the CCP4 graphical interface ccp4i and points out some issues that need to be considered, together with a few examples. It covers detn. of the point-group symmetry of the diffraction data (the Laue group), which is required for the subsequent scaling step, examn. of systematic absences, which in many cases will allow inference of the space group, putting multiple data sets on a common indexing system when there are alternatives, the scaling step itself, which produces a large set of data-quality indicators, estn. of |F| from intensity and finally examn. of intensity statistics to detect crystal pathologies such as twinning. An appendix outlines the scoring schemes used by the program POINTLESS to assign probabilities to possible Laue and space groups.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktFWqtLo%253D&md5=f425388a87744d721d324118d50e4f9a
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Winn, M. D. ; Ballard, C. C. ; Cowtan, K. D. ; Dodson, E. J. ; Emsley, P. ; Evans, P. R. ; Keegan, R. M. ; Krissinel, E. B. ; Leslie, A. G. ; McCoy, A. ; McNicholas, S. J. ; Murshudov, G. N. ; Pannu, N. S. ; Potterton, E. A. ; Powell, H. R. ; Read, R. J. ; Vagin, A. ; Wilson, K. S. Overview of the CCP4 suite and current developments. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67 , 235– 242, DOI: 10.1107/S0907444910045749

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52

Overview of the CCP4 suite and current developments

Winn, Martyn D.; Ballard, Charles C.; Cowtan, Kevin D.; Dodson, Eleanor J.; Emsley, Paul; Evans, Phil R.; Keegan, Ronan M.; Krissinel, Eugene B.; Leslie, Andrew G. W.; McCoy, Airlie; McNicholas, Stuart J.; Murshudov, Garib N.; Pannu, Navraj S.; Potterton, Elizabeth A.; Powell, Harold R.; Read, Randy J.; Vagin, Alexei; Wilson, Keith S.

Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 235-242CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)

A review. The CCP4 (Collaborative Computational Project, No. 4) software suite is a collection of programs and assocd. data and software libraries which can be used for macromol. structure detn. by X-ray crystallog. The suite is designed to be flexible, allowing users a no. of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by std. file formats, data objects and graphical interfaces. Structure soln. by macromol. crystallog. is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktFWqt70%253D&md5=c407e4d47bef46864be336d60147c17d
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Vagin, A. ; Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 , 22– 25, DOI: 10.1107/S0907444909042589

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53

Molecular replacement with MOLREP

Vagin, Alexei; Teplyakov, Alexei

Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (1), 22-25CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)

MOLREP is an automated program for mol. replacement that utilizes a no. of original approaches to rotational and translational search and data prepn. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron d., structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calcd. from the input data.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXit1Kktw%253D%253D&md5=820d114719aca209994ffb0403e3b20d
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Lebedev, A. A. ; Young, P. ; Isupov, M. N. ; Moroz, O. V. ; Vagin, A. A. ; Murshudov, G. N. JLigand: a graphical tool for the CCP4 template-restraint library. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2012, 68 , 431– 440, DOI: 10.1107/S090744491200251X

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54

JLigand: a graphical tool for the CCP4 template-restraint library

Lebedev, Andrey A.; Young, Paul; Isupov, Michail N.; Moroz, Olga V.; Vagin, Alexey A.; Murshudov, Garib N.

Acta Crystallographica, Section D: Biological Crystallography (2012), 68 (4), 431-440CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)

Biol. macromols. are polymers and therefore the restraints for macromol. refinement can be subdivided into two sets: restraints that are applied to atoms that all belong to the same monomer and restraints that are assocd. with the covalent bonds between monomers. The CCP4 template-restraint library contains three types of data entries defining template restraints: descriptions of monomers and their modifications, both used for intramonomer restraints, and descriptions of links for intermonomer restraints. The library provides generic descriptions of modifications and links for protein, DNA and RNA chains, and for some post-translational modifications including glycosylation. Structure-specific template restraints can be defined in a user's addnl. restraint library. Here, JLigand, a new CCP4 graphical interface to LibCheck and REFMAC that has been developed to manage the user's library and generate new monomer entries is described, as well as new entries for links and assocd. modifications.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xlt1Gnsr4%253D&md5=333c42dac336b0598540500f06350d3a
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Emsley, P. ; Lohkamp, B. ; Scott, W. G. ; Cowtan, K. Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66 , 486– 501, DOI: 10.1107/S0907444910007493

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55

Features and development of Coot

Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.

Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)

Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFKisb8%253D&md5=67262cbfc60004de5ef962d5c043c910
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Murshudov, G. N. ; Vagin, A. A. ; Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53 , 240– 255, DOI: 10.1107/S0907444996012255

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56

Refinement of macromolecular structures by the maximum-likelihood method

Murshudov, Garib N.; Vagin, Alexei A.; Dodson, Eleanor J.

Acta Crystallographica, Section D: Biological Crystallography (1997), D53 (3), 240-255CODEN: ABCRE6; ISSN:0907-4449. (Munksgaard)

A review with many refs. on the math. basis of max. likelihood. The likelihood function for macromol. structures is extended to include prior phase information and exptl. std. uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estg. σA using "free" reflections is described and its effects analyzed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial α-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the max.-likelihood residual are consistently better than those obtained from least-squares refinement.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjs1Gnsb4%253D&md5=ec7f141ce1542f7ff458b98ecfe3f8af
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Murshudov, G. N. ; Skubak, P. ; Lebedev, A. A. ; Pannu, N. S. ; Steiner, R. A. ; Nicholls, R. A. ; Winn, M. D. ; Long, F. ; Vagin, A. A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67 , 355– 367, DOI: 10.1107/S0907444911001314

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57

REFMAC5 for the refinement of macromolecular crystal structures

Murshudov, Garib N.; Skubak, Pavol; Lebedev, Andrey A.; Pannu, Navraj S.; Steiner, Roberto A.; Nicholls, Robert A.; Winn, Martyn D.; Long, Fei; Vagin, Alexei A.

Acta Crystallographica, Section D: Biological Crystallography (2011), 67 (4), 355-367CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)

This paper describes various components of the macromol. crystallog. refinement program REFMAC5, which is distributed as part of the CCP4 suite. REFMAC5 utilizes different likelihood functions depending on the diffraction data employed (amplitudes or intensities), the presence of twinning and the availability of SAD/SIRAS exptl. diffraction data. To ensure chem. and structural integrity of the refined model, REFMAC5 offers several classes of restraints and choices of model parameterization. Reliable models at resolns. at least as low as 4 Å can be achieved thanks to low-resoln. refinement tools such as secondary-structure restraints, restraints to known homologous structures, automatic global and local NCS restraints, 'jelly-body' restraints and the use of novel long-range restraints on at. displacement parameters (ADPs) based on the Kullback-Leibler divergence. REFMAC5 addnl. offers TLS parameterization and, when high-resoln. data are available, fast refinement of anisotropic ADPs. Refinement in the presence of twinning is performed in a fully automated fashion. REFMAC5 is a flexible and highly optimized refinement package that is ideally suited for refinement across the entire resoln. spectrum encountered in macromol. crystallog.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXktFWqtbk%253D&md5=f8f3202d246908500057ad7c71015b7b
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Nakabayashi, H. ; Taketa, K. ; Miyano, K. ; Yamane, T. ; Sato, J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982, 42 , 3858– 3863

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58

Growth of human hepatoma cell lines with differentiated functions in chemically defined medium

Nakabayashi, Hidekazu; Taketa, Kazuhisa; Miyano, Keiko; Yamane, Takashi; Sato, Jiro

Cancer Research (1982), 42 (9), 3858-63CODEN: CNREA8; ISSN:0008-5472.

A human hepatoma cell line, HuH-7, which was established from a hepatocellular carcinoma, replicated continuously in a chem. defined medium when the medium was supplemented with Na2SeO3. The cells grew better in this medium than in serum-contg. medium without any adaptation period. Other established human hepatoma and hepatoblastoma cell lines, HuH-6 cl-5, PLC/PRF/5, huH-1, and huH-4, also grew in the defined medium. Although HLEC-1 cells failed to proliferate continuously with Na2SeO3 alone, they grew if a cell-free conditioned medium from HuH-7 cells was added to the medium. These cell lines, except the HLEC-1 cell line, produced the following human plasma proteins: albumin, prealbumin, α1-antitrypsin, ceruloplasmin, fibrinogen, fibronectin, haptoglobin, hemopexin, β-lipoprotein, α2-macroglobulin, β2-microglobulin, transferrin, complement components 3 and 4, and α1-fetoprotein. Beside plasma proteins, the media from HuH-7, HuH-6 cl-5, PLC/PRF/5, and huH-1 contained anticarcinoembryonic antigen-reactive proteins, and those from PLC/PRF/5, huH-1, and huH-4 medium contained hepatitis B surface antigen. These proteins were detected during periods of serial cultivation over 9 mo under the above culture conditions. The hepatoma cell lines grown in the fully defined synthetic medium may provide a new approach for investigating the growth and metab. of human hepatoma cells in vitro.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38Xlt1ens78%253D&md5=ea9da2cd2c0090618a3c2e5d924799e0
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Schultz, D. E. ; Honda, M. ; Whetter, L. E. ; McKnight, K. L. ; Lemon, S. M. Mutations within the 5′ nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells. J. Virol. 1996, 70 , 1041– 1049, DOI: 10.1128/JVI.70.2.1041-1049.1996

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59

Mutations within the 5' nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells

Schultz, Derk E.; Honda, Masao; Whetter, Linda E.; McKnight, Kevin L.; Lemon, Stanley M.

Journal of Virology (1996), 70 (2), 1041-9CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)

Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell culture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in cultured African green monkey kidney (BS-C-1) cells but not in fetal rhesus monkey kidney (FRhK-4) cells. To det. whether these mutations enhance cap-independent translation directed by the HAV internal ribosomal entry site (IRES), the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses were compared in 2 stably transformed cell lines (BT7-H and FRhK-T7) which constitutively express cytoplasmic bacteriophage T7 RNA polymerase and which are derived from BS-C-1 and FRhK-4 cells, resp. Translational activity was assessed by monitoring expression of a reporter protein, chloramphenicol acetyltransferase (CAT), following transfection with plasmid DNAs contg. bicistronic T7 transcriptional units of the form luciferase-5'NTR-CAT. In both cell types, transcripts contg. the 5'NTR of HM175/P16 expressed CAT at levels that were 50-100-fold lower than transcripts contg. the IRES elements of Sabin type 1 poliovirus or encephalomyocarditis virus, confirming the low activity of the HAV IRES. However, in BT7-H cells, transcripts contg. the 5'NTR of HM175/P16 expressed CAT with 4-5-fold greater efficiency than transcripts contg. the 5'NTR of wild-type virus. This translational enhancement was due to additive effects of a UU deletion at nucleotides 203 and 204 and a U-to-G substitution at nucleotide 687 of HM175/P16. These mutations did not enhance translation in FRhK-T7 or Huh-T7 cells (a T7 polymerase-expressing cell line derived from human hepatoblastoma cells) or in vitro in rabbit reticulocyte lysates. These results demonstrate that mutations in the 5'NTR of a cell culture-adapted HAV enhance viral replication by facilitating cap-independent translation in a cell-type-specific fashion and support the concept that picornaviral host range is detd. in part by differences in cellular translation initiation factors.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xks1aitg%253D%253D&md5=d412d80343840753354df4052fbb8148
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Pfefferle, S. ; Schöpf, J. ; Kögl, M. ; Friedel, C. C. ; Müller, M. A. ; Carbajo-Lozoya, J. ; Stellberger, T. ; von Dall'Armi, E. ; Herzog, P. ; Kallies, S. ; Niemeyer, D. ; Ditt, V. ; Kuri, T. ; Züst, R. ; Pumpor, K. ; Hilgenfeld, R. ; Schwarz, F. ; Zimmer, R. ; Steffen, I. ; Weber, F. ; Thiel, V. ; Herrler, G. ; Thiel, H. J. ; Schwegmann-Wessels, C. ; Pöhlmann, S. ; Haas, J. ; Drosten, C. ; von Brunn, A. The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors. PLoS Pathog. 2011, 7 , e1002331, DOI: 10.1371/journal.ppat.1002331

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60

The SARS-Coronavirus-host interactome: identification of cyclophilins as target for Pan-Coronavirus inhibitors

Pfefferle, Susanne; Schoepf, Julia; Koegl, Manfred; Friedel, Caroline C.; Mueller, Marcel A.; Carbajo-Lozoya, Javier; Stellberger, Thorsten; von Dall'Armi, Ekatarina; Herzog, Petra; Kallies, Stefan; Niemeyer, Daniela; Ditt, Vanessa; Kuri, Thomas; Zuest, Roland; Pumpor, Ksenia; Hilgenfeld, Rolf; Schwarz, Frank; Zimmer, Ralf; Steffen, Imke; Weber, Friedemann; Thiel, Volker; Herrler, Georg; Thiel, Heinz-Juergen; Schwegmann-Wessels, Christel; Poehlmann, Stefan; Haas, Juergen; Drosten, Christian; von Brunn, Albrecht

PLoS Pathogens (2011), 7 (10), e1002331CODEN: PPLACN; ISSN:1553-7374. (Public Library of Science)

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurol. disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biol. approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These mols. modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as obsd. in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivs. of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhsVyqurnN&md5=345bb753d8c4af3bad86ea6ba0f1099b
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de Wilde, A. H. ; Raj, V. S. ; Oudshoorn, D. ; Bestebroer, T. M. ; van Nieuwkoop, S. ; Limpens, R. W. ; Posthuma, C. C. ; van der Meer, Y. ; Barcena, M. ; Haagmans, B. L. ; Snijder, E. J. ; van den Hoogen, B. G. MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-alpha treatment. J. Gen. Virol. 2013, 94 , 1749– 1760, DOI: 10.1099/vir.0.052910-0

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61

MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment

de Wilde, Adriaan H.; Raj, V. Stalin; Oudshoorn, Diede; Bestebroer, Theo M.; van Nieuwkoop, Stefan; Limpens, Ronald W. A. L.; Posthuma, Clara C.; van der Meer, Yvonne; Barcena, Montserrat; Haagmans, Bart L.; Snijder, Eric J.; van den Hoogen, Bernadette G.

Journal of General Virology (2013), 94 (8), 1749-1760CODEN: JGVIAY; ISSN:0022-1317. (Society for General Microbiology)

Coronavirus (CoV) infections are commonly assocd. with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. The authors characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, the authors obsd. rapidly increasing viral RNA synthesis and release of high titers of infectious progeny, followed by a pronounced cytopathol. These characteristics were used to develop an assay for antiviral compd. screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV is 50-100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht12gs7rI&md5=f252b101487365f030a6613d7c5b7c82
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Snijder, E. J. ; van der Meer, Y. ; Zevenhoven-Dobbe, J. ; Onderwater, J. J. ; van der Meulen, J. ; Koerten, H. K. ; Mommaas, A. M. Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex. J. Virol. 2006, 80 , 5927– 5940, DOI: 10.1128/JVI.02501-05

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62

Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex

Snijder, Eric J.; van der Meer, Yvonne; Zevenhoven-Dobbe, Jessika; Onderwater, Jos J. M.; van der Meulen, Jannes; Koerten, Henk K.; Mommaas, A. Mieke

Journal of Virology (2006), 80 (12), 5927-5940CODEN: JOVIAM; ISSN:0022-538X. (American Society for Microbiology)

The RNA replication complexes of mammalian pos.-stranded RNA viruses are generally assocd. with (modified) intracellular membranes, a feature thought to be important for creating an environment suitable for viral RNA synthesis, recruitment of host components, and possibly evasion of host defense mechanisms. Here, using a panel of replicase-specific antisera, we have analyzed the earlier stages of severe acute respiratory syndrome coronavirus (SARS-CoV) infection in Vero E6 cells, in particular focusing on the subcellular localization of the replicase and the ultrastructure of the assocd. membranes. Confocal immunofluorescence microscopy demonstrated the colocalization, throughout infection, of replicase cleavage products contg. different key enzymes for SARS-CoV replication. Electron microscopy revealed the early formation and accumulation of typical double-membrane vesicles, which probably carry the viral replication complex. The vesicles appear to be fragile, and their preservation was significantly improved by using cryofixation protocols and freeze substitution methods. In immunoelectron microscopy, the virus-induced vesicles could be labeled with replicase-specific antibodies. Opposite to what was described for mouse hepatitis virus, we did not observe the late relocalization of specific replicase subunits to the presumed site of virus assembly, which was labeled using an antiserum against the viral membrane protein. This conclusion was further supported using organelle-specific marker proteins and electron microscopy. Similar morphol. studies and labeling expts. argued against the previously proposed involvement of the autophagic pathway as the source for the vesicles with which the replicase is assocd. and instead suggested the endoplasmic reticulum to be the most likely donor of the membranes that carry the SARS-CoV replication complex.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XlsFWmsL4%253D&md5=c30e126fb9412ac2ad9dc3e62870ef99
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de Wilde, A. H. ; Jochmans, D. ; Posthuma, C. C. ; Zevenhoven-Dobbe, J. C. ; van Nieuwkoop, S. ; Bestebroer, T. M. ; van den Hoogen, B. G. ; Neyts, J. ; Snijder, E. J. Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture. Antimicrob. Agents Chemother. 2014, 58 , 4875– 4884, DOI: 10.1128/AAC.03011-14

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63

Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture

de Wilde, Adriaan H.; Jochmans, Dirk; Posthuma, Clara C.; Zevenhoven-Dobbe, Jessika C.; van Nieuwkoop, Stefan; Bestebroer, Theo M.; van den Hoogen, Bernadette G.; Neyts, Johan; Snijder, Eric J.

Antimicrobial Agents and Chemotherapy (2014), 58 (8), 4875-4884, 11 pp.CODEN: AMACCQ; ISSN:1098-6596. (American Society for Microbiology)

Coronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar previously unknown coronavirus emerged, Middle East respiratory syndrome coronavirus (MERS-CoV), thus far causing over 650 lab.-confirmed infections, with an unexplained steep rise in the no. of cases being recorded over recent months. The human MERS fatality rate of ∼30% is alarmingly high, even though many deaths were assocd. with underlying medical conditions. Registered therapeutics for the treatment of coronavirus infections are not available. Moreover, the pace of drug development and registration for human use is generally incompatible with strategies to combat emerging infectious diseases. Therefore, we have screened a library of 348 FDA-approved drugs for anti-MERS-CoV activity in cell culture. If such compds. proved sufficiently potent, their efficacy might be directly assessed in MERS patients. We identified four compds. (chloroquine, chlorpromazine, loperamide, and lopinavir) inhibiting MERS-CoV replication in the low-micromolar range (50% effective concns. [EC50s], 3 to 8 μM). Moreover, these compds. also inhibit the replication of SARS coronavirus and human coronavirus 229E. Although their protective activity (alone or in combination) remains to be assessed in animal models, our findings may offer a starting point for treatment of patients infected with zoonotic coronaviruses like MERS-CoV. Although they may not necessarily reduce viral replication to very low levels, a moderate viral load redn. may create a window during which to mount a protective immune response.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhs1artrfJ&md5=3fac7c18295ca0aca41632cbb9bd1280
64

Chan, J. F. ; Choi, G. K. ; Tsang, A. K. ; Tee, K. M. ; Lam, H. Y. ; Yip, C. C. ; To, K. K. ; Cheng, V. C. ; Yeung, M. L. ; Lau, S. K. ; Woo, P. C. ; Chan, K. H. ; Tang, B. S. ; Yuen, K. Y. Development and evaluation of novel real-time reverse transcription-PCR assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses. J. Clin. Microbiol. 2015, 53 , 2722– 2726, DOI: 10.1128/JCM.01224-15

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64

Development and evaluation of novel real-time reverse transcription-PCR assays with locked nucleic acid probes targeting leader sequences of human-pathogenic coronaviruses

Chan, Jasper Fuk-Woo; Choi, Garnet Kwan-Yue; Tsang, Alan Ka-Lun; Tee, Kah-Meng; Lam, Ho-Yin; Yip, Cyril Chik-Yan; To, Kelvin Kai-Wang; Cheng, Vincent Chi-Chung; Yeung, Man-Lung; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Chan, Kwok-Hung; Tang, Bone Siu-Fai; Yuen, Kwok-Yung

Journal of Clinical Microbiology (2015), 53 (8), 2722-2726CODEN: JCMIDW; ISSN:1098-660X. (American Society for Microbiology)

Based on findings in small RNA-sequencing (Seq) data anal., we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Anal. and clin. evaluations showed their noninferiority to a com. multiplex PCR test for the detection of these coronaviruses.

https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVCitbzJ&md5=6278c3e165cf4ec572eddeab12978187
65

Carbajo-Lozoya, J. ; Ma-Lauer, Y. ; Malesevic, M. ; Theuerkorn, M. ; Kahlert, V. ; Prell, E. ; von Brunn, B. ; Muth, D. ; Baumert, T. F. ; Drosten, C. ; Fischer, G. ; von Brunn, A. Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including Alisporivir. Virus Res. 2014, 184 , 44– 53, DOI: 10.1016/j.virusres.2014.02.010

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65

Human coronavirus NL63 replication is cyclophilin A-dependent and inhibited by non-immunosuppressive cyclosporine A-derivatives including Alisporivir

Carbajo-Lozoya, Javier; Ma-Lauer, Yue; Malesevic, Miroslav; Theuerkorn, Martin; Kahlert, Viktoria; Prell, Erik; von Brunn, Brigitte; Muth, Doreen; Baumert, Thomas F.; Drosten, Christian; Fischer, Gunter; von Brunn, Albrecht

Virus Research (2014), 184 (), 44-53CODEN: VIREDF; ISSN:0168-1702. (Elsevier B.V.)

Until recently, there were no effective drugs available blocking coronavirus (CoV) infection in humans and animals. We have shown before that CsA and FK506 inhibit coronavirus replication (Carbajo-Lozoya, J., Mueller, M.A., Kallies, S., Thiel, V., Drosten, C., von Brunn, A. Replication of human coronaviruses SARS-CoV, HCoV-NL63 and HCoV-229E is inhibited by the drug FK506. Virus Res. 2012; Pfefferle, S., Schoepf, J., Koegl, M., Friedel, C., Mueller, M.A., Stellberger, T., von Dall'Armi, E., Herzog, P., Kallies, S., Niemeyer, D., Ditt, V., Kuri, T., Zuest, R., Schwarz, F., Zimmer, R., Steffen, I., Weber, F., Thiel, V., Herrler, G., Thiel, H.-J., Schwegmann-Wessels, C., Poehlmann, S., Haas, J., Drosten, C. and von Brunn, A. The SARS-Coronavirus-host interactome: identification of cyclophilins as target for pan-Coronavirus inhibitors. PLoS Pathog., 2011). Here we demonstrate that CsD Alisporivir, NIM811 as well as novel non-immunosuppressive derivs. of CsA and FK506 strongly inhibit the growth of human coronavirus HCoV-NL63 at low micromolar, non-cytotoxic concns. in cell culture. We show by qPCR anal. that virus replication is diminished up to four orders of magnitude to background levels. Knockdown of the cellular Cyclophilin A (CypA/PPIA) gene in Caco-2 cells prevents replication of HCoV-NL63, suggesting that CypA is required for virus replication. Collectively, our results uncover Cyclophilin A as a host target for CoV infection and provide new strategies for urgently needed therapeutic approaches.

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Note

Introduction

Figure 1

Results

Structure-Based Design of α-Ketoamides

Scheme 1

Synthesis of α-Ketoamides

Viral Replicons

Initial Inhibitor Design Steps

Figure 2

Figure 3

P1′ and P3 Substituents

Properties of the S2 Pockets of the Target Enzymes

P2-Alkyl Substituents of Varying Sizes

Modifying Ring Size and Flexibility of P2-Cycloalkylmethyl Substituents

Figure 4

Discussion

Conclusions

Experimental Section

Crystallization and X-ray Structure Determination of Complexes between Viral Proteases and α-Ketoamides

Crystallization

Diffraction Data Collection, Structure Elucidation, and Refinement

Inhibitory Activity Assay of α-Ketoamides

Cells and Viruses

Viral Replicons

Transfection

Testing for Inhibitory Activity of Candidate Compounds

Antiviral Assay with Infectious Enteroviruses

Antiviral Assays with SARS and MERS Coronaviruses

Antiviral Assay with Human Coronavirus 229E

Determination of the Cell Toxicity of Candidate Compounds

Chemical Synthesis of α-Ketoamides

General Procedure

Synthesis of (2S,4R)-Dimethyl 2-(tert-butoxycarbonylamino)-4-(cyanomethyl)pentanedioate (1)

Synthesis of (S)-Methyl 2-(tert-butoxycarbonylamino)-3-((S)-2-oxopyrrolidin-3-yl)propanoate (2)

Synthesis of (S)-Methyl 2-Amino-3-((S)-2-oxopyrrolidin-3-yl)propanoate (3)

Synthesis of Methyl N-Substituted Amino-acid Esters 4

General Procedure

(S)-Methyl 2-Cinnamamido-3-phenylpropanoate (4a)

Synthesis of N-Substituted Amino Acids 5 (General Procedure)

Synthesis of Compounds 6 (General Procedure)

Synthesis of Alcohols 7 (General Procedure)

Synthesis of Aldehydes 8 (General Procedure)

Synthesis of Compounds 9 (General Procedure)

Synthesis of α-Hydroxyamides 10 (General Procedure)

Synthesis of α-Ketoamides 11 (General Procedure)

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-phenylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11a):

tert-Butyl ((S)-4-(Benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)carbamate (11f)

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-(4-fluorophenyl)propanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11m):

(S)-N-((S)-4-(Benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)-2-cinnamamido-4-methylpentanamide (11n):

(S)-N-((S)-4-(Benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)-2-cinnamamidohexanamide (11o):

(S)-N-((S)-4-(Benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)-2-cinnamamidopent-4-ynamide (11p):

(S)-N-Benzyl-3-((S)-2-cinnamamido-2-cyclopropylacetamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11q):

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-cyclohexylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11r):

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-cyclopropylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11s):

(S)-N-benzyl-3-((S)-2-cinnamamido-3-cyclobutylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11t):

(S)-N-Benzyl-3-((S)-2-cinnamamido-3-cyclopentylpropanamido)-2-oxo-4-((S)-2-oxopyrrolidin-3-yl)butanamide (11u):

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