Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors

Abstract

A new coronavirus (CoV) identified as COVID-19 virus is the etiological agent responsible for the 2019-2020 viral pneumonia outbreak that commenced in Wuhan [1][2][3][4] . Currently there are no targeted therapeutics and effective treatment options remain very limited. In order to rapidly discover lead compounds for clinical use, we initiated a program of combined structure-assisted drug design, virtual drug screening and high-throughput screening to identify new drug leads that target the COVID-19 virus main protease (M pro ). M pro is a key CoV enzyme, which plays a pivotal role in mediating viral replication and transcription, making it an attractive drug target for this virus 5,6 . Here, we identified a mechanism-based inhibitor, N3, by computer-aided drug design and subsequently determined the crystal structure of COVID-19 virus M pro in complex with this compound. Next, through a combination of structure-based virtual and high-throughput screening, we assayed over 10,000 compounds including approved drugs, drug candidates in clinical trials, and other pharmacologically active compounds as inhibitors of M pro . Six of these compounds inhibited M pro with IC 50 values ranging from 0.67 to 21.4 μM. Ebselen also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of this screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.CoVs infect humans and other animal species, causing a variety of highly prevalent and severe diseases, including Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) 7 . The COVID-19 virus genome is comprised of ~30,000 nucleotides; its replicase gene encodes two overlapping polyproteins, pp1a and pp1ab, required for viral replication and transcription 3,4 . The functional polypeptides are released from the polyproteins by extensive proteolytic processing, predominantly by a 33.8-kDa main protease (M pro ), also referred to as the 3C-like protease. M pro digests the polyprotein at no less than 11 conserved sites, starting with the autolytic cleavage of this enzyme itself from pp1a and pp1ab 8 . The functional importance of M pro in the viral life cycle, together with the absence of closely related homologues in humans, identify the M pro as an attractive target for antiviral drug design 9 .To facilitate the rapid discovery of antiviral compounds with clinical potential, we developed a strategy combining structure-assisted drug design, virtual drug screening and high-throughput screening to repurpose existing drugs to target COVID-19 virus M pro . Establishing a high-throughput activity assayRecombinant COVID-19 virus M pro with native N and C termini was expressed in Escherichia coli and subsequently purified (Extended Data Fig. 1a, b). The molecular weight of COVID-19 virus M pro as determined by mass spectroscopy is 33797.0 Da, consistent with its theoretical molecular weight 337...

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

“…The compounds identified in this study represent the most potent and selective hits reported so far, and are superior than recently reported SARS-CoV-2 M pro inhibitors ebselen, N3, and 13b (Table 5). Aside from the above positive results, we also showed that ritonavir (9) and lopinavir (10) failed to inhibit the SARS-CoV-2 M pro (IC50 > 20 µM, Fig. 2), which might explain their lack efficacy in clinical trials for COVID-19.…”

“…28 As the SARS-CoV-2 M pro shares a high sequence similarity with SARS and to a less extent with MERS, we reasoned that inhibiting the enzymatic activity of SARS-CoV-2 M pro will similarly prevent viral replication. 7,9 Noticeable findings from our study include: 1) Boceprevir (28) or aldehyde prodrug, the bisulfite (GC-376 (64)). This result suggests that reactive warheads might be essential for SARS-CoV-2 M pro inhibition.…”

“…The protease inhibitors are grouped based on their targets and mechanism of action and include proteasome inhibitors (1-8); HIV protease inhibitors (9)(10)(11)(12)(13)(14); γ-secretase inhibitors (15)(16)(17)(18)(19)(20)(21)(22); HCV NS3-4A protease inhibitors (23)(24)(25)(26)(27)(28)(29); DPP-4 inhibitors (30)(31)(32)(33)(34)(35); miscellaneous serine protease inhibitors (36)(37)(38)(39); cathepsin and calpain protease inhibitors (40-43); miscellaneous cysteine protease inhibitors (44-48); matrix metalloprotease inhibitors (49-51); and miscellaneous protease inhibitors (52-55). The inhibitors were pre-incubated with 100 nM of M pro at 30 °C for 30 minutes in the presence of 4 mM 1,4-dithiothreitol (DTT) before the addition of 10 µM FRET substrate.…”

“…by digesting the viral polyproteins at more than 11 sites, and it appears like a high profile target for antiviral drug discovery. [7][8][9] The M pro has an unique substrate preference for glutamine at the P1 site (Leu-Gln(Ser,Ala,Gly)), a feature that is absent in closely related host proteases, suggesting it is feasible to achieve high selectivity by targeting viral M pro . As such, we developed the Fluorescence Resonance Energy Transfer (FRET)-based enzymatic assay for the SARS-CoV-2 M pro and applied it to screen a focused library of protease inhibitors.…”

“…The compounds identified herein represent the most potent and selective SARS-CoV-2 M pro inhibitors so far with both enzymatic inhibition and cellular antiviral activity. 7,9…”