Aug 2026· Drug Discoveries & Therapeutics· 0 citations· 45 references
Medicine
TL;DR
The findings provide mechanistic insights into a key allosteric mechanism for Mpro inhibition but also provide a promising chemical scaffold for further development as an Mpro-targeting inhibitor.
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a crucial therapeutic target for anti-coronavirus disease 2019 (COVID-19) drug development, as it is essential for viral replication. However, mutations within the active site have compromised the efficacy of current competitive inhibitors, prompting the exploration of alternative inhibition strategies. In this study, we systematically investigated the allosteric inhibition mechanism of SARS-CoV-2 Mpro by pelitinib and leveraged this insight for new inhibitor discovery. Through extensive molecular dynamics simulations, we showed that pelitinib exerts allosteric inhibition via the L141-S144-C145-H41 interaction network: it restricts the flexibility of L141 through CH-π interactions, transmits this effect to C145 via S144, stabilizes the hydrogen bond between C145 and H41, and thereby reduces the flexibility of the S3 helix (residues 40-60). This series of conformational changes induces the contraction of the Mpro catalytic pocket from ~1200 ų to ~800 ų, impairs substrate binding, and ultimately appears to impair Mpro activity. Based on this mechanism, we performed structure-based virtual screening and identified a novel compound (Cpd-1). Biological evaluations showed that Cpd-1 exhibits superior Mpro inhibitory activity compared to pelitinib, with negligible off-target binding to human EGFR and Myt1 kinase, low cytotoxicity (cell viability > 60% at 200 μM), and predicted inhibitory activity against clinically relevant Mpro-resistant mutants based on computational analysis. Our findings provide mechanistic insights into a key allosteric mechanism for Mpro inhibition but also provide a promising chemical scaffold for further development as an Mpro-targeting inhibitor.
Findings establish that both catalytic and allosteric sites play essential roles in regulating 3CL pro function, and targeting allosteric regions such as the Asn28-associated pocket offers a promising approach for antiviral development.
The SARS-CoV-2 main protease (3CLpro) is an essential enzyme for viral replication and a major target for antiviral drug development. While its catalytic activity is known to require dimer formation, the mechanism by which it cleaves itself from the monomeric viral polyprotein remains to be further studied. In this study, we engineered several D-peptides based on a de novo designed D-peptide inhibitor. These peptides bind to the 3CLpro monomer and unexpectedly activate its catalytic function. By enhancing the turnover number, the peptides significantly boost the catalytic efficiency of 3CLpro. Among them, LY11 stands out with high binding affinity (KD = 117 nM) and strong potency of enzymatic activation (EC200 < 1 μM). Through biophysical and computational approaches, we show that LY11 binds in 1:1 stoichiometry, stabilizing the monomeric state while allosterically remodeling the inter-domain linker of 3CLpro to expand the substrate binding pocket and switch on the protease. These findings suggest new insights into the maturation and catalytic mechanism of 3CLpro. The LY11-regulated 3CL protease may serve as a versatile molecular tool for synthetic biology applications.
Lai-Yi Feng, Xin-Liao Ling, Weijie Bian et al.· Protein Science· 0 citations
The continuous evolution of SARS-CoV-2 and the emergence of drug-resistant variants underscore the urgent need for broad-spectrum antiviral agents targeting conserved viral proteins. The main protease (Mpro) represents a promising target due to its essential role in coronavirus replication. In this study, we report the discovery and optimization of a novel series of piperazine-based Mpro inhibitors using a multi-site binding strategy guided by analysis of conserved residues within the coronavirus Mpro active sites. Starting from the noncovalent lead GC-14, systematic optimization of substituents occupying the S1', S1, S2, and S4 subsites of Mpro led to the development of the noncovalent inhibitor GY-e2, which showed improved inhibitory efficacy against both SARS-CoV-2 and SARS-CoV Mpro. To further enhance its antiviral efficacy in cellular models, reactive warheads targeting C145 were incorporated into the scaffold to generate covalent inhibitors. This strategy yielded the isomeric compounds Y-U0-R and Y-U0-S, which displayed potent Mpro inhibition and markedly enhanced antiviral activity in SARS-CoV-2-infected Calu-3 cells. Moreover, both compounds exhibited broad-spectrum antiviral activity against other human coronaviruses, and notably remained effective against the two major Nirmatrelvir-resistant strains evaluated in this study. Mechanistic studies further confirmed kinetically stable binding and time-dependent inhibition of Y-U0-R, supporting the rationale of covalent inhibitor design. These findings highlight the utility of structure-based design for the development of promising broad-spectrum anti-coronavirus agents.
Bing Ye, M. Lee, Letian Song et al.· European journal of medicina...· 0 citations
This study provides binding details for the designed compounds and demonstrates the feasibility of the joint X-ray/neutron structure-assisted drug design approach to generate more potent noncovalent nonpeptidic SARS-CoV-2 MPro inhibitors.
Dipendra Bhandari, Katerina Kovalevskaya, L. Coates et al.· RSC Medicinal Chemistry· 0 citations
New SARS-CoV-2 Mpro small-molecule inhibitors endowed with a pyrimidine scaffold are designed and synthesized and the mechanism of action of the most promising compound was elucidated.
Salvatore Nieddu, Giuseppe Ruggieri, Riccardo De Santis et al.· ACS Infectious Diseases· 0 citations
The SARS-CoV-2 main protease (MPro) is an essential enzyme for viral replication and a primary target for antiviral drug development. Despite extensive structural and biochemical characterization, the allosteric mechanisms by which dimerization informs conformational changes at active site lack an explicit comparison across the different states that identify key residues that connect substrate binding, dimerization, and catalytic activation. Here, we integrate microsecond time scale all-atom molecular dynamics (MD) simulations with dynamical network analysis to characterize how ligand binding and dimerization modulate the allosteric communication landscape of MPro. We performed triplicate 1-μs simulations of MPro in the monomer and dimer states. For each of these states, we simulated MPro in the apo state, as well as bound to a natural peptide substrate (nsp 15/16), the covalent inhibitor nirmatrelvir (Paxlovid) and the noncovalent inhibitor ensitrelvir (Xocova). Dynamical network analyses from the resulting simulations reveal that dimerization redirects the highest correlated motions from the interdomain loop towards the domain II and III interface. At the dimer interface, we identified N-terminal and domain II β-hairpin residues that act as central communication hubs creating networks that connect both chains in the dimer. Small molecule binding to the active site further modulates these networks in distinct ways: nirmatrelvir and peptide substrate binding results in the formation of allosteric networks within the oxyanion loop, while ensitrelvir-bound monomeric MPro results in a dimer-like network, suggesting an inhibitory "allosteric switch" mechanism that may hinder dimerization upon binding. Across all systems, domain III emerges as an allosteric "pivot", providing a platform that allows the most relevant networks to connect inter-chain communication to the active site upon dimerization. Together, these findings define how correlated motion networks couple active-site dynamics to dimerization and ligand binding, providing molecular insight into the principles governing allosteric regulation in MPro. This framework highlights potential avenues for developing antivirals that target not only the catalytic site but also the communication pathways sustaining dimer stability and enzymatic function.
Javier O. Sanlley Hernandez, Carla Calvó-Tusell, Fiona L. Kearns et al.· Biophysical Journal· 0 citations
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