SARS-CoV-2 Mpro inhibitors and activity-based probes for patient-sample imaging

Rut, Wioletta; Groborz, Katarzyna; Zhang, Linlin; Sun, Xinyuanyuan; Żmudziński, Mikołaj; Pawlik, Bartłomiej; Wang, Xinyu; Jochmans, Dirk; Neyts, Johan; Młynarski, Wojciech; Hilgenfeld, Rolf; Drąg, Marcin

doi:10.1038/s41589-020-00689-z

Cited by 274 publications

(392 citation statements)

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“…In conclusion, inhibition of the M pro and PL pro proteases is considered to be a potentially activity in cells 26,37 . In this work, we describe our efforts to screen a library of approximately 650 diverse covalent inhibitor scaffolds against the two primary SARS-CoV-2 cysteine proteases, M pro and PL pro .…”

Section: Resultsmentioning

confidence: 90%

“…To identify potential inhibitors of M pro and PL pro , we developed fluorogenic substrate assays that allowed us to screen a focused library of cysteine reactive molecules. We based the design of internally quenched-fluorescent M pro substrates on recent specificity profiling of the P1-4 residues using non-natural amino acids with a C-terminal 7-amino-4-carbamoylmethylcoumarin (ACC) reporter 26 . However, because the reported structures have relatively low turnover rates, we decided to make extended versions of these substrates that combine the optimal P1-4 residues with the native cleavage consensus of the P1'-P3' residues (i.e., residues C-terminal of the scissile bond) 26,27 .…”

Section: Resultsmentioning

confidence: 99%

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Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

Steuten

Kim

Widen

et al. 2020

Preprint

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Two proteases produced by the SARS-CoV-2 virus, Mpro and PLpro, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsin L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

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Section: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

Steuten

Kim

Widen

et al. 2020

Preprint

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“…The benzyl ring of the α-methylbenzyl moiety is partially positioned both in the S2 and S3 pockets, a novel binding pose that has not been observed with existing M pro inhibitors. Normally, a substituent at this position would be expected to flip away from the enzyme core towards the solvent-exposed S3 pocket, which explains why P3 substitutions have little to no influence on the enzymatic inhibition 4 . However, the hydrophobic nature of the benzyl ring in 23R causes it to project towards the core near the S2 pocket, forcing Gln189 to rotate outwards (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…M pro is a cysteine protease encoded in the viral polyprotein as non-structural protein 5 (Nsp5) that cleaves the viral polyproteins pp1a and pp1ab at more than 11 sites. Despite its multiple proteolytic sites, M pro was shown to have a high substrate specificity of glutamine at the P1 position 4 . As such, the majority of the reported M pro inhibitors were designed to contain a 2-pyrrolidone at the P1 substitution as a mimetic of the glutamine in the substrate 5 .…”

mentioning

confidence: 99%

An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity

Kitamura

Sacco

et al. 2020

Preprint

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The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II and XII, each containing a reactive warhead that covalently modifies the catalytic Cys145. In this study, we report an expedited drug discovery approach by coupling structure-based design and Ugi four-component (Ugi-4CR) reaction methodology to the design of non-covalent Mpro inhibitors. The most potent compound 23R had cellular antiviral activity similar to covalent inhibitors such as GC376. Our designs were guided by overlaying the structure of SARS-CoV Mpro + ML188 (R), a non-covalent inhibitor derived from Ug-4CR, with the X-ray crystal structures of SARS-CoV-2 Mpro + calpain inhibitor XII/GC376/UAWJ247. Binding site analysis suggests a strategy of extending the P2 and P3 substitutions in ML188 (R) to achieve optimal shape complementary with SARS-CoV-2 Mpro. Lead optimization led to the discovery of 23R, which inhibits SARS-CoV-2 Mpro and SARS-CoV-2 viral replication with an IC50 of 0.31 μM and EC50 of 1.27 μM, respectively. The binding and specificity of 23R to SARS-CoV-2 Mpro were confirmed in a thermal shift assay and native mass spectrometry assay. The co-crystal structure of SARS-CoV-2 Mpro with 23R revealed the P2 biphenyl fits snuggly into the S2 pocket and the benzyl group in the α-methylbenzyl faces towards the core of the enzyme, occupying a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study revealed the most potent non-covalent SARS-CoV-2 Mpro inhibitors reported to date and a novel binding pocket that can be explored for Mpro inhibitor design.

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“…1e), speci cally, Leu27, Asn119, and Gly146 are near the catalytic dyad (His41 and Cys145). They may provide some support to the catalytic center, as evidenced by a recent study, in which Leu27 was found to play a key role in the activity of the M pro structure of SARS-CoV2 7 . Whereas, Leu27 and Asn119 are involved in the formation of the binding site in SARS-CoV M pro 16 (Table 1).…”

mentioning

confidence: 92%

Identification of SARS-CoV2 Main Protease Coldspots Suitable for Drug Targeting

Krishnamoorthy

Fakhro

2020

Preprint

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Most attempts to target the novel coronavirus SARS-CoV2 are focusing on the main protease (Mpro) 1,2. We already have access to high resolution 3D-structures of the SARS-CoV2 Mpro, which were developed with inhibitors as co-crystals using X-ray crystallography 3-9. However, >19,000 missense mutations in the Mpro have already been reported 10. The mutations encompassing 282 amino acid positions and these “hotspots” might change the Mpro structure and activity, potentially rendering novel antivirals and vaccines ineffective. Here we identified 24 mutational “coldspots” that have resisted mutation since the virus was first detected. We compared the structure-function relationship of these coldspots with several SARS-CoV2 Mpro X-ray crystal structures. We found that three coldspot residues (Leu141, Phe185 and Gln192) help to form the active site, while six (Gly2, Arg4, Tyr126, Lys137, Leu141 and Leu286) contribute to dimer formation that is required for Mpro activity. Importantly, seven coldpots are conserved among other coronaviruses and available on the surface of the active site and at the dimer interface for targeting. The identification and short list of these coldspots offers a new perspective to target the SARS-CoV2 Mpro while avoiding mutation-based drug resistance.

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SARS-CoV-2 Mpro inhibitors and activity-based probes for patient-sample imaging

Abstract: Substrate specificity of SARS-CoV and SARS-CoV-2 M pro s. To determine the SARS-CoV M pro and SARS-CoV-2 M pro substrate preferences, we applied a HyCoSuL approach. The library consists of

Cited by 274 publications

References 36 publications

Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

Challenges for targeting SARS-CoV-2 proteases as a therapeutic strategy for COVID-19

An expedited approach towards the rationale design of non-covalent SARS-CoV-2 main protease inhibitors with in vitro antiviral activity

Identification of SARS-CoV2 Main Protease Coldspots Suitable for Drug Targeting

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