Aug 2026· RSC Medicinal Chemistry· 0 citations· 102 references
Medicine
TL;DR
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.
Abstract
SARS-CoV-2 main protease (MPro) is a proven target for drug discovery of small-molecule antiviral agents due to its crucial role in viral polyprotein processing, high structural conservation across numerous divergent variants, and the lack of similar human enzymes. Unlike covalent compounds, noncovalent inhibitors of MPro do not modify the enzyme's active site, and may offer improved safety profiles, greater chemical tractability, and better oral bioavailability without the need for pharmacokinetic enhancement. In this study, we designed, synthesized and characterized thirteen noncovalent nonpeptidic SARS-CoV-2 MPro inhibitors clustered into two series (KK and KB) of compounds. The inhibitors were designed based on our recently discovered Mcule-5948770040 and its analogue HL-3-68 designed through the structure–activity relationship study. To obtain atomic details of the inhibitors' binding we solved room-temperature X-ray structures of the MPro/inhibitor complexes, and to quantify their binding and antiviral properties we performed in vitro DSF and ITC measurements and TCID50 antiviral assays. In addition, a room-temperature neutron structure of the MPro/KB-5 complex allowed direct determination of hydrogen positions, mapping intermolecular interactions and directly visualizing the protonation states and hydrogen bonding. Improved binding affinities of KK-7 and KB-3 through KB-6 could be attributed to the observed nonconventional S–H⋯F hydrogen bond and an additional conventional hydrogen bond between the carboxamide moieties and Q189. Our study provides binding details for the designed compounds and demonstrates the feasibility of our joint X-ray/neutron structure-assisted drug design approach to generate more potent noncovalent nonpeptidic MPro inhibitors.
The integrated rational design-based synthesis, biological evaluation, and computational investigations collectively identified thiadiazole/oxadiazole scaffolds as promising candidates for the development of SARS-CoV-2 inhibitors, offering valuable insights for next-generation antiviral agents targeting coronavirus proteases.
Rafaqat Hussain, Hina Sarfraz, T. Chohan et al.· Pure and Applied Chemistry· 0 citations
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 main protease (Mpro) is a prime target for anti-SARS-CoV-2 drugs. Herein, we describe the structure-based covalent modifications that generate new nonpeptidic covalent Mpro inhibitors. The compounds exhibit strong inhibition against Mpro at submicromolar levels. The deuterated lead compound exhibits submicromolar antiviral activity against wild-type SARS-CoV-2 and Omicron variants. Biolayer interferometry analysis indicates a strong binding affinity for Mpro. The mass spectrometry results confirm the covalent bonding of the compound with the cysteine residue of Mpro. Taken together, the data may support the deuterated compound as a new lead for developing anti-SARS-CoV-2 drugs and further reveal the feasibility of using chloroacetyl for structure-based covalent drug design.
Honglei Bao, Ling-Chen Sun, Shilin Gong et al.· ACS Bio & Med Chem Au· 0 citations
Developing covalent inhibitors of the SARS-CoV-2 papain-like protease (PLpro) represents a promising therapeutic strategy, but translation of this approach into in vivo antiviral efficacy remains limited. A major challenge is the shallow and minimally druggable binding environment surrounding the catalytic cysteine, which reflects the enzyme's stringent requirement for P1 and P2 Gly-Gly recognition. In addition, many previously reported covalent PLpro inhibitors have relied on warheads or linker designs with limited metabolic stability or nonspecific thiol reactivity, restricting their progression toward in vivo efficacy. Here, we report the design and synthesis of a novel class of covalent PLpro inhibitors with low intrinsic GSH reactivity, enabled by a chloroalkyne warhead and an optimized linker strategy. The lead compound, ID3-77 (12), potently inhibits PLpro biochemically, demonstrates strong cellular antiviral activity, reduces viral load in a SARS-CoV-2 infection model, and exhibits minimal glutathione labeling. Jump-dilution experiments demonstrate reversible covalent inhibition, while profiling against the cysteine proteases cathepsin B and calpain demonstrates selectivity over these related proteases. Together, these findings establish chloroalkyne as a promising target-directed reversible covalent warhead for PLpro inhibition and provide in vivo proof-of-concept for this chemistry in antiviral drug discovery, with potential for broader application to other cysteine proteases.
Divakar Indukuri, J. G. Achi, Boopathi Sownthirarajan et al.· Bioorganic & Medicinal Chemi...· 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
The SARS-CoV-2 main protease (Mpro) and host cysteine protease cathepsin L (CatL) are both attractive targets for antiviral intervention. Herein we describe the rational design and synthesis of dual Mpro/CatL inhibitors derived from the previously reported leads SM141 and SM142. Optimization focused on replacement of the acrylate ester warhead in SM141 and SM142 with a nitrile, modifying the P2, P3, and P4 capping groups, and variation of the P1 lactam ring size. In addition, conformational restriction was achieved through macrocyclization between the P1 and P4 side chains to enhance binding affinity. These efforts identified multiple dual-target inhibitors with nanomolar potency against both Mpro and CatL and potent antiviral activity as well as selective Mpro inhibitors with strong enzymatic activity. Targeting both a viral protease and a host factor may provide more durable antiviral efficacy by reducing susceptibility to resistance arising from viral evolution of Mpro.
Leonard Barasa, Yong-Zhi Chen, Ala M. Shaqra et al.· ACS Medicinal Chemistry Lett...· 0 citations
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