Promising antiviral protease inhibitors With no vaccine or proven effective drug against the virus that causes coronavirus disease 2019 (COVID-19), scientists are racing to find clinical antiviral treatments. A promising drug target is the viral main protease Mpro, which plays a key role in viral replication and transcription. Dai et al. designed two inhibitors, 11a and 11b, based on analyzing the structure of the Mpro active site. Both strongly inhibited the activity of Mpro and showed good antiviral activity in cell culture. Compound 11a had better pharmacokinetic properties and low toxicity when tested in mice and dogs, suggesting that this compound is a promising drug candidate. Science, this issue p. 1331

Abstract SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the etiological agent responsible for the global COVID-19 (coronavirus disease 2019) outbreak. The main protease of SARS-CoV-2, Mpro, is a key enzyme that plays a pivotal role in mediating viral replication and transcription. We designed and synthesized two lead compounds (11a and 11b) targeting Mpro. Both exhibited excellent inhibitory activity and potent anti–SARS-CoV-2 infection activity. The x-ray crystal structures of SARS-CoV-2 Mpro in complex with 11a or 11b, both determined at a resolution of 1.5 angstroms, showed that the aldehyde groups of 11a and 11b are covalently bound to cysteine 145 of Mpro. Both compounds showed good pharmacokinetic properties in vivo, and 11a also exhibited low toxicity, which suggests that these compounds are promising drug candidates.

In late December 2019, a cluster of pneumonia cases caused by a novel coronavirus was reported in Wuhan, China (1–3). Genomic sequencing showed that this pathogenic coronavirus is 96% identical to a bat coronavirus and shares 79.6% sequence identity to SARS-CoV (4–6). This novel coronavirus was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses, and the pneumonia was designated as coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO) on 11 February 2020 (7). The epidemic spread rapidly to more than 212 countries and was announced as a global health emergency by WHO (8). No clinically effective vaccines or specific antiviral drugs are currently available for the prevention and treatment of COVID-19 infections. The combination of α-interferon and the anti-HIV drugs lopinavir/ritonavir (Kaletra) has been used, but the curative effect remains very limited and there can be toxic side effects (9). Remdesivir, a broad-spectrum antiviral drug developed by Gilead Sciences Inc., is also being explored for the treatment of COVID-19, but more data are needed to prove its efficacy (10–12). Specific anti–SARS-CoV-2 drugs offering efficacy and safety are urgently needed.

A maximum likelihood tree based on the genomic sequence showed that the virus falls within the subgenus Sarbecovirus of the genus Betacoronavirus (6). Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses. The genomic RNA of coronaviruses is ~30,000 nucleotides in length with a 5′-cap structure and a 3′-poly(A) tail, and contains at least six open reading frames (ORFs) (13, 14). The first ORF (ORF 1a/b), about two-thirds of the genome length, directly translates two polyproteins, pp1a and pp1ab, so named because there is an a-1 frameshift between ORF1a and ORF1b. These polyproteins are processed by a main protease, Mpro [also known as the 3C-like protease (3CLpro)], and by one or two papain-like proteases, into 16 nonstructural proteins (NSPs). These NSPs engage in the production of subgenomic RNAs that encode four main structural proteins [envelope (E), membrane (M), spike (S), and nucleocapsid (N) proteins] and other accessory proteins (15, 16). Therefore, these proteases, especially Mpro, play a vital role in the life cycle of coronaviruses.

Mpro is a three-domain (domains I to III) cysteine protease involved in most maturation cleavage events within the precursor polyprotein (17–19). Active Mpro is a homodimer containing two protomers. The coronavirus Mpro features a noncanonical Cys-His dyad located in the cleft between domains I and II (17–19). Mpro is conserved among coronaviruses, and several common features are shared among the substrates of Mpro in different coronaviruses. The amino acids in substrates from the N terminus to the C terminus are numbered as follows: -P4-P3-P2-P1↓P1′-P2′-P3′-, with the cleavage site between P1 and P1′. In particular, a Gln is almost always required in the P1 position of the substrates. Because Mpro has no human homolog, it is an ideal antiviral target (20–22).

Design and synthesis of 11a and 11b The active sites of Mpro are highly conserved among all coronavirus Mpros and are usually composed of four sites: S1′, S1, S2, and S4 (22). We were able to design and synthesize inhibitors targeting SARS-CoV-2 Mpro by analyzing the substrate-binding pocket of SARS-CoV Mpro (PDB ID 2H2Z) (Fig. 1). The thiol of a cysteine residue in the S1′ site anchors inhibitors by a covalent linkage that is important for the inhibitors to maintain antiviral activity. In our design of new inhibitors, an aldehyde was selected as a new warhead in P1 in order to form a covalent bond with cysteine. The reported SARS-CoV Mpro inhibitors often have an (S)-γ-lactam ring that occupies the S1 site of Mpro, and this ring was expected to be a good choice in P1 (23). Furthermore, the S2 site of coronavirus Mpro is usually large enough to accommodate the larger P2 fragment. To test the importance of different ring systems, we introduced a cyclohexyl or 3-fluorophenyl into P2, with the fluorine expected to enhance activity. An indole group was introduced into P3 to form new hydrogen bonds with S4 and improve drug-like properties. Fig. 1 Design strategy of SARS-CoV-2 main protease (Mpro) inhibitors and the chemical structures of 11a and 11b. The synthetic route and chemical structures of the compounds (11a and 11b) are shown in scheme S1. The starting material (N-Boc-l-glutamic acid dimethyl ester 1) was obtained from commercial suppliers and used without further purification to synthesize the key intermediate 3 according to the literature (24). The intermediates 6a and 6b were synthesized from 4 and acids 5a and 5b. Removal of the t-butoxycarbonyl group from 6a and 6b yielded 7a and 7b. Coupling 7a and 7b with the acid 8 yielded the esters 9a and 9b. The peptidomimetic aldehydes 11a and 11b were approached through a two-step route in which the ester derivatives 9 were first reduced with NaBH 4 to generate the primary alcohols 10a and 10b, which were subsequently oxidized into aldehydes 11a and 11b with Dess-Martin periodinane (DMP).

11a and 11b are potent inhibitors of Mpro Recombinant SARS-CoV-2 Mpro was expressed and purified from Escherichia coli (18, 25). A fluorescently labeled substrate—MCA-AVLQ↓SGFR-Lys(Dnp)-Lys-NH 2 , derived from the N-terminal autocleavage sequence from the viral protease—was designed and synthesized for the enzymatic assay. Both 11a and 11b exhibited high SARS-CoV-2 Mpro inhibition activity, which reached 100% for 11a and 96% for 11b at 1 μM, respectively. We used a fluorescence resonance energy transfer (FRET)–based cleavage assay to determine the median inhibitory concentration (IC 50 ) values. The results revealed excellent inhibitory potency, with IC 50 values of 0.053 ± 0.005 μM and 0.040 ± 0.002 μM for 11a and 11b, respectively (Fig. 2). Fig. 2 Inhibitory activity profiles of compounds 11a (A) and 11b (B) against SARS-CoV-2 Mpro.

Structures of SARS-CoV-2 Mpro in complex with 11a and 11b To elucidate the mechanism of inhibition of SARS-CoV-2 Mpro by 11a, we determined the crystal structure of this complex at 1.5 Å resolution (table S1). The crystal of Mpro-11a belongs to the space group C2, and an asymmetric unit contains only one molecule (table S1). Two molecules (designated protomer A and protomer B) associate into a homodimer around a crystallographic two-fold symmetry axis (fig. S2). The structure of each protomer contains three domains with the substrate-binding site located in the cleft between domains I and II. At the active site of SARS-CoV-2 Mpro, Cys145 and His41 (Cys-His) form a catalytic dyad (fig. S2). The electron density map clearly shows compound 11a in the substrate-binding pocket of SARS-CoV-2 Mpro in an extended conformation (Fig. 3A and fig. S3, A and B). Details of the interaction are shown in Fig. 3, B and C. The electron density shows that the C of the aldehyde group of 11a and the catalytic-site Cys145 of SARS-CoV-2 Mpro form a standard 1.8 Å C–S covalent bond. The oxygen atom of the aldehyde group also plays a crucial role in stabilizing the conformations of the inhibitor by forming a 2.9 Å hydrogen bond with the backbone of residue Cys145 in the S1′ site. The (S)-γ-lactam ring of 11a at P1 fits well into the S1 site. The oxygen of the (S)-γ-lactam group forms a 2.7 Å hydrogen bond with the side chain of His163. The main chain of Phe140 and side chain of Glu166 also participate in stabilizing the (S)-γ-lactam ring by forming 3.2 Å and 3.0 Å hydrogen bonds with its NH group, respectively. In addition, the amide bonds on the chain of 11a form hydrogen bonds with the main chains of His164 (3.2 Å) and Glu166 (2.8 Å), respectively. The cyclohexyl moiety of 11a at P2 deeply inserts into the S2 site, stacking with the imidazole ring of His41. The cyclohexyl group is also surrounded by the side chains of Met49, Tyr54, Met165, Asp187, and Arg188, producing extensive hydrophobic interactions. The indole group of 11a at P3 is exposed to solvent (S4 site) and is stabilized by Glu166 through a 2.6 Å hydrogen bond. The side chains of residues Pro168 and Gln189 interact with the indole group of 11a through hydrophobic interactions. Multiple water molecules (named W1 to W6) play an important role in binding 11a. W1 interacts with the amide bonds of 11a through a 2.9 Å hydrogen bond, whereas W2 to W6 contribute to stabilizing 11a in the binding pocket by forming a number of hydrogen bonds with the aldehyde group of 11a and the residues of Asn142, Gly143, Thr26, Thr25, His41, and Cys44. Fig. 3 Mpro-inhibitor binding modes for 11a and 11b. (A) Cartoon representation of the crystal structure of SARS-CoV-2 Mpro in complex with 11a. Compound 11a is shown as magenta sticks; water molecules are shown as red spheres. (B) Close-up view of the 11a binding pocket. Four subsites, S1′, S1, S2, and S4, are labeled. The residues involved in inhibitor binding are shown as wheat sticks. 11a and water molecules are shown as magenta sticks and red spheres, respectively. Hydrogen bonds are indicated as dashed lines. (C) Schematic diagram of SARS-CoV-2 Mpro–11a interactions shown in (B). (D) Comparison of the binding modes of 11a and 11b in SARS-CoV-2 Mpro. The major difference between 11a and 11b is marked with a dashed circle; 11a and 11b are shown as magenta and yellow sticks, respectively. (E) Close-up view of the 11b binding pocket. Hydrogen bonds are indicated as dashed lines. (F) Schematic diagram of SARS-CoV-2 Mpro–11b interactions shown in (E). Amino acid abbreviations: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. The crystal structure of SARS-CoV-2 Mpro in complex with 11b is very similar to that of the 11a complex and shows a similar inhibitor-binding mode (Fig. 3D and figs. S3, C and D, and S4A). The difference in binding mode is most probably due to the 3-fluorophenyl group of 11b at P2. Relative to the cyclohexyl group in 11a, the 3-fluorophenyl group undergoes a downward rotation (Fig. 3D). The side chains of residues His41, Met49, Met165, Val186, Asp187, and Arg188 interact with this aryl group through hydrophobic interactions, and the side chain of Gln189 stabilizes the 3-fluorophenyl group with an additional 3.0 Å hydrogen bond (Fig. 3, E and F). In short, these two crystal structures reveal a similar inhibitory mechanism in which both compounds occupy the substrate-binding pocket and block the enzyme activity of SARS-CoV-2 Mpro. Compounds N1, N3, and N9 are wide-spectrum inhibitors targeting coronavirus Mpro. Compared with the binding modes of N1, N3, and N9 in SARS-CoV Mpro complex structures reported previously, the binding modes of 11a and 11b in SARS-CoV-2 Mpro complex structures are similar and the differences among these overall structures are small (Fig. 4 and fig. S4, B to F) (22). The differences mainly lie in the interactions at S1′, S2, and S4 subsites, possibly because the sizes of functional groups vary at the corresponding P1′, P2, and P4 sites in the inhibitors (Fig. 4, A and C). Fig. 4 Comparison of the inhibitor binding modes in SARS-CoV Mpro and SARS-CoV-2 Mpro. (A) Comparison of binding modes of 11a in SARS-CoV-2 Mpro with those of N1, N3, and N9 in SARS-CoV Mpro. SARS-CoV-2 Mpro–11a (wheat; PDB code 6LZE), SARS-CoV Mpro–N1 (sky blue; PDB code 1WOF), SARS-CoV Mpro–N3 (gray; PDB code 2AMQ), and SARS-CoV Mpro–N9 (olive; PDB code 2AMD) are shown as cartoons. 11a, N1, N3, and N9 are shown in magenta, cyan, dirty violet, and salt, respectively. (B) Comparison of the 11a and N3 binding pockets. Residues in the Mpro-11a and Mpro-N3 structures are colored in wheat and gray, respectively. 11a and N3 are shown as sticks colored in magenta and dirty violet, respectively. (C) Comparison of binding modes of 11b in SARS-CoV-2 Mpro with those of N1, N3, and N9 in SARS-CoV Mpro. SARS-CoV-2 Mpro–11b (PDB code 6M0K) is shown as a pale cyan cartoon. 11b, N1, N3, and N9 are shown in yellow, cyan, dirty violet, and salt, respectively. (D) Comparison of the 11b and N9 binding pockets. Residues in the Mpro-11b and Mpro-N9 structures are colored in pale cyan and olive, respectively. 11b and N9 are shown as sticks colored in yellow and salt, respectively.

Antiviral activity of 11a and 11b To further substantiate the enzyme inhibition results, we evaluated the ability of these compounds to inhibit SARS-CoV-2 in vitro (Fig. 5 and fig. S5). As shown in Fig. 5, compounds 11a and 11b exhibited good anti–SARS-CoV-2 infection activity in cell culture, with half-maximal effective concentration (EC 50 ) values of 0.53 ± 0.01 μM and 0.72 ± 0.09 μM, respectively, by plaque assay. Neither compound caused cytotoxicity, with half cytotoxic concentration (CC 50 ) values of >100 μM, yielding selectivity indices for 11a and 11b of >189 and >139, respectively. We also used immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) to monitor the antiviral activity of 11a and 11b. The results showed that 11a and 11b exhibit a good antiviral effect on SARS-CoV-2 (Fig. 5 and fig. S5). Fig. 5 In vitro inhibition of viral main protease inhibitors against SARS-CoV-2. (A and B) Vero E6 cells were treated with a series concentration of indicated compounds 11a and 11b and infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.05. At 24 hours after infection, viral yield in the cell supernatant was quantified by plaque assay. The cytotoxicity of these compounds in Vero E6 cells was also determined by using CCK8 assays. The left and right y axis of each graph represents mean percent inhibition of virus yield and mean percent cell viability of the drugs, respectively. (C and D) Viral RNA copy numbers in the cell supernatants were quantified by qRT-PCR. Data are means ± SD; n = 3 biological replicates.

Pharmacokinetic and toxicity studies To explore the further druggability of the compounds 11a and 11b, we evaluated both compounds for their pharmacokinetic properties. As shown in table S2, compound 11a given to mice intraperitoneally (5 mg/kg) and intravenously (5 mg/kg) displayed a half-life (T 1/2 ) of 4.27 hours and 4.41 hours, respectively, and we observed a high maximal concentration (C max = 2394 ng/ml) and a good bioavailability of 87.8% when the compound 11a was given intraperitoneally. Metabolic stability of 11a in mice was also good (clearance = 17.4 ml min–1 kg–1). When administered intraperitoneally (20 mg/kg), subcutaneously (5 mg/kg), and intravenously (5 mg/kg), compound 11b also showed good pharmacokinetic properties (its bioavailability exceeded 80% when given both intraperitoneally and subcutaneously, and it displayed a longer T 1/2 of 5.21 hours when given intraperitoneally). Considering the danger of COVID-19, we selected intravenous drip administration for further study because of its comparatively high area under the curve (AUC) value and rapid effect. Relative to 11a administrated intravenously in CD-1 mice, 11b displayed a shorter T 1/2 (1.65 hours) and a faster clearance rate (clearance = 20.6 ml min–1 kg–1). Compound 11a was selected for further investigation with intravenous drip dosing in Sprague-Dawley (SD) rats and beagle dogs. The results showed (table S3) that 11a exhibited long T 1/2 values (SD rat, 7.6 hours; beagle dog, 5.5 hours), low clearance rates (rat, 4.01 ml min–1 kg–1; dog, 5.8 ml min–1 kg–1), and high AUC values (rat, 41,500 hours·ng/ml; dog, 14,900 hours·ng/ml). The above pharmacokinetic results indicate that compound 11a warrants further study. An in vivo toxicity study (table S4) of 11a was carried out on SD rats and beagle dogs. The acute toxicity of 11a was measured in SD rats. No SD rats died after receiving 40 mg/kg by intravenous drip administration. When the dosage was raised to 60 mg/kg, one of four SD rats died. The dose range toxicity of 11a was studied for 7 days at dosing levels of 2, 6, and 18 mg/kg in SD rats and at 10 to 40 mg/kg in beagle dogs. All animals received once-daily (QD) dosing by intravenous drip, and all animals were clinically observed at least once a day. No obvious toxicity was observed in either group. These data indicate that 11a is a good candidate for further clinical study.

Supplementary Materials science.sciencemag.org/content/368/6497/1331/suppl/DC1 Materials and Methods Scheme S1 Figs. S1 to S5 Tables S1 to S4 References (26–29)

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