SARS-CoV-2 Mpro inhibitors with antiviral activity in a transgenic mouse model

Targeting the SARS-CoV-2 main protease Vaccines are an important tool in the fight against COVID-19, but developing antiviral drugs is also a high priority, especially with the rise of variants that may partially evade vaccines. The viral protein main protease is required for cleaving precursor polyproteins into functional viral proteins. This essential function makes it a key drug target. Qiao et al. designed 32 inhibitors based on either boceprevir or telaprevir, both of which are protease inhibitors approved to treat hepatitis C virus. Six compounds protected cells from viral infection with high potency, and two of these were selected for in vivo studies based on pharmokinetic experiments. Both showed strong antiviral activity in a mouse model. Science, this issue p. 1374

T he COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1-3). Despite intensive countermeasures implemented around the world, morbidity and mortality remain high with many countries facing a new wave of infections (4,5). Because limited antiviral agents are available to combat SARS-CoV-2 infection, the development of specific antiviral drugs against SARS-CoV-2 is urgently needed.
SARS-CoV-2 is an enveloped positive-sense single-stranded RNA virus belonging to the genus Betacoronavirus (1)(2)(3)6). This virus contains a~30-kb RNA genome encoding two large overlapping polyprotein precursors (pp1a and pp1ab), four structural proteins (spike, envelope, membrane, and nucleocapsid), and several accessory proteins (1,2,6). The cleavage of the two polyproteins (pp1a/pp1ab) into individual nonstructural proteins is essential for viral genome replication. This cleaving process is performed by two viral proteases: main protease (M pro , also named 3CL protease) and papain-like protease (7). These viral proteases are thus important antiviral targets (8,9). Notably, M pro exclusively cleaves polypeptides after a glutamine (Gln) residue, and no known human protease displays the same cleavage specificity as M pro (9,10). This may allow the development of drugs that are specific to M pro to reduce potential side effects.
The design of SARS-CoV-2 M pro inhibitors was based on the reported crystal structures of SARS-CoV-2 M pro (11)(12)(13) and our cocrystal structures of SARS-CoV-2 M pro in complex with the approved antivirals against hepatitis C virus infection, boceprevir (PDB entry 7COM) and telaprevir (PDB entry 7C7P) ( fig. S1). The active site of M pro is composed of four sites (S1′, S1, S2, and S4), which often accommodate four fragments (P1′, P1, P2, and P3, respectively) of peptidomimetic inhibitors (8,10). In our design of new inhibitors (Fig. 1), we fixed P1 as an optimal fragment, used P2 that was derived from either boceprevir or telaprevir, and allowed P3 to change. First, an aldehyde was used as the warhead in P1 to form a covalent bond with the catalytic site Cys 145 , which is essential for the antiviral activity (13). Relative to other bulky warheads, the small and highly electrophilic aldehyde has been reported to be more potent (7,10,20,22). However, the clinical safety of the generated aldehydes remains to be determined because of possible off-target effects due to the high electrophilicity of aldehyde (23). Second, we chose a five-membered ring (g-lactam) derivative of glutamine to occupy the S1 site of M pro , which not only mimics the native P1 glutamine of the substrates but also increases the activity of inhibitors (24,25). Third, we used a bicycloproline moiety, either (1R,2S,5S)-6,6-dimethyl-3-aza-bicyclo[3.1.0] hexane-2-formamide (P2 of boceprevir) or (1S,3aR,6aS)-octahydrocyclopenta[c]pyrrole-1formamide (P2 of telaprevir), as a P2 fragment. This was inspired by our cocrystal structures of SARS-CoV-2 M pro in complex with boceprevir and telaprevir (fig. S1), in which the two bicycloproline moieties suitably occupy the S2 pocket of M pro . In particular, the rigid and hydrophobic bicycloproline is expected to increase drug exposure in vivo (26). Finally, by analyzing the characteristics of the S4 site of M pro , we decided to use hydrophobic subgroups of medium size for P3 to enhance the potency and pharmacokinetic (PK) properties of the resulting inhibitors. We thus designed and synthesized 32 compounds with various P3 fragments (MI-01 to MI-32; fig. S2 routes, and characterization of these compounds by nuclear magnetic resonance and electrospray ionization mass spectrometry.] The 32 compounds' biochemical activities against SARS-CoV-2 M pro were determined by a fluorescence resonance energy transfer (FRET) assay. For this, recombinant SARS-CoV-2 M pro protein was prepared. The turnover number (k cat )/Michaelis constant (K m ) value of the recombinant protein was determined as 50,656 ± 4221 M -1 s -1 , similar to a previous result (11). In the FRET assay, all 32 compounds (MI-01 to MI-32) showed potent inhibitory activities on SARS-CoV-2 M pro , with 50% inhibitory concentration (IC 50 ) values ranging from 7.6 to 748.5 nM (table S1). Of these, 24 compounds displayed two-digit nanomolar IC 50 values, and three exhibited single-digit values (MI-21, 7.6 nM; MI-23, 7.6 nM; MI-28, 9.2 nM). The positive controls GC376 and 11b, two of the most potent SARS-CoV-2 M pro inhibitors reported (13,17), exhibited IC 50 values of 37.4 nM and 27.4 nM in the same assay, respectively. Next, a differential scanning fluorimetry (DSF) assay was performed to validate the direct binding between our compounds and SARS-CoV-2 M pro . All the compounds displayed large thermal shifts ranging from 12.5°to 21.7°C (table S1), indicating their tight binding to SARS-CoV-2 M pro . It is noteworthy that the two different bicycloproline moieties (P2) did not affect the inhibitory activities and binding abilities (e.g., MI-03 versus MI-21, MI-12 versus MI-28, and MI-14 versus MI-30; table S1 and fig. S2).
To illustrate the detailed binding mode of our compounds with SARS-CoV-2 M pro , we determined the 2.0-Å structure of M pro in complex with one of the most active compounds, MI-23 (IC 50 = 7.6 nM) (Fig. 2, A to D). The crystal structure of the M pro -MI-23 complex belongs to space group C2 (table S2) with one molecule per asymmetric unit. The biological dimer of M pro is formed by an M pro monomer and its symmetry-mate across the crystallographic two-fold axis ( Fig. 2A). MI-23 binds  to the active site of M pro as expected (Fig. 2, C  and D). The carbon of the warhead aldehyde interacts with the sulfur atom of catalytic residue Cys 145 to form a 1.8-Å covalent bond (Fig. 2C). The oxygen of the aldehyde forms two hydrogen bonds with the main-chain amides of Cys 145 and Gly 143 (forming the "oxyanion hole") (Fig. 2D). The P1 g-lactam ring of MI-23 inserts deeply into the S1 pocket.  (Fig. 2D). The benzene ring of P3 also forms a very weak hydrophobic interaction with Gly 251 from an adjacent translational symmetry protomer as a result of crystal packing. Overall, the binding pattern of the representative compound MI-23 with M pro is consistent with our design concept.
We then selected 20 compounds with IC 50 < 50 nM in the enzyme inhibition assay to examine their cytotoxicity and cellular antiviral activity. First, the cytotoxicity of these compounds was evaluated using the Cell Counting Kit-8 (CCK8) assay (Beyotime Biotechnology), and no compounds showed cytotoxicity [half cytotoxic concentration (CC 50 ] > 500 mM] in the cell lines tested, including Vero E6, HPAEpiC, LO2, BEAS-2B, A549, and Huh7 cells (tables S3 and S4).
Next, the compounds' cellular antiviral activity was examined by a cell protection assay. In this assay, the viability of SARS-CoV-2infected Vero E6 cells with or without treatment with the compounds was assessed using CCK8. All the compounds dose-dependently protected cells from death with 50% effective concentration (EC 50 ) values ranging from 0.53 to 30.49 mM (table S4) (Fig. 3A). We noticed that some compounds (e.g., MI-22 and MI-25) with high potency in the enzymatic assay showed marginal activity in the cell protection assay, perhaps due to relatively low lipophilic groups in P3 and the resulting poor cell membrane permeability (28). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) revealed that all six compounds inhibited SARS-CoV-2 virus replication in HPAEpiC cells with low-nanomolar EC 50 values (0.3 to 7.3 nM) (Fig. 3B). In the same CCK8 and RT-qPCR assays, the positive control GC376 showed EC 50 values of 1.46 mM and 153.1 nM, respectively, and the corresponding values for 11b were 0.89 mM and 23.7 nM. To further corroborate the antiviral potency of these compounds, we conducted RT-qPCR in another cell line, Huh7. The six compounds showed antiviral EC 50 values of 31.0 to 96.7 nM, whereas GC376 and 11b displayed EC 50 values of 174.9 nM and 74.5 nM, respectively ( fig. S5).
To identify which of the six compounds is suitable for in vivo antiviral studies, we conducted PK experiments in Sprague-Dawley rats. Two compounds, MI-09 and MI-30, showed relatively good PK properties with oral bioavailability of 11.2% and 14.6%, respectively (table S5). Because a compound with oral bioavailability of >10% has potential for development as an oral drug (29), MI-09 and MI-30 were selected for further in vivo antiviral study.
The key PK parameters of MI-09 and MI-30 are summarized in Fig. 4, A (table S6). In a repeated dose toxicity study, treatment with MI-09 or MI-30 by i.v. at 6 and 18 mg kg -1 day -1 , i.p. at 100 and 200 mg kg -1 day -1 , or p.o. at 100 and 200 mg/kg twice daily for 7 consecutive days did not result in noticeable toxicity in the animals (table S6).
Further, we investigated the in vivo antiviral activity of our compounds in a human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model, which is susceptible to SARS-CoV-2 (30). In our pilot study, hACE2 transgenic mice were intranasally inoculated 4 of 5 with SARS-CoV-2 [2 × 10 6 TCID 50 (50% tissue culture infectious dose) virus per mouse] and were then treated with vehicle (control), MI-09 [50 mg/kg p.o. twice daily (bid) or 50 mg/kg i.p. once daily (qd)] or MI-30 (50 mg/kg i.p. qd) starting at 1 hour prior to virus inoculation (Fig. 4C) and continuing until 5 days postinfection (5 dpi). During the 6-day period, no abnormal behaviors or body weight loss were observed in any animals tested. At 1 dpi, the mean viral RNA loads in the lung tissues of the three treatment groups were significantly (P < 0.05, Student's t test) lower than that of the control group (Fig. 4D). At 3 and 5 dpi, the viral RNA loads in the lung tissues of treatment groups were almost undetectable, and those of the control group were also very low [below the limit of detection (LOD)], which might be due to the mild degree of infection.
We thus increased the virus challenge dose of SARS-CoV-2 to 5 × 10 6 TCID 50 , which mimics a moderate infection. The mice were treated as described above, except that the doses increased to 100 mg/kg for both i.p. and p.o. administration of MI-09 and MI-30 (Fig. 4C). The higher dose of virus challenge led to a higher level of viral loads in the lungs of infected mice, as expected. The mean viral RNA loads in the lung tissues of the three treatment groups were slightly lower than those of the control group at 1 dpi and significantly lower (P < 0.05, Student's t test) at 3 dpi (Fig.  4D). At 5 dpi, the viral loads in the lung tissues were undetectable in the treatment groups and were low (near or below LOD) in the control group.
Histopathological analysis was performed for the lungs of mice infected with SARS-CoV-2 at 5 × 10 6 TCID 50 . At 3 dpi, the vehicletreated mice showed moderate alveolar septal thickening and inflammatory cell infiltration, whereas all compound-treated animals exhibited slight alveolar septal thickening and mild inflammatory cell infiltration (Fig. 4E). To investigate whether the compounds ameliorate lung damage by affecting host immune response, we studied the expression of inflam-matory cytokines and chemokines as well as immune cell infiltration in the lungs. MI-09 or MI-30 reduced the expression levels of IFN-b and CXCL10 (Fig. 4F). Also, fewer neutrophils and macrophages occurred in the lungs of compound-treated mice than in control mice (Fig. 4, G and H), suggesting inhibition of immune cell infiltration. Together, our results show that i.p. or p.o. administration of MI-09 or MI-30 could efficiently inhibit SARS-CoV-2 replication and ameliorate SARS-CoV-2-induced lung lesions in vivo, and they represent an important step toward the development of orally available anti-SARS-CoV-2 drugs.