Aug 2026· ACS Infectious Diseases· Vol 12 9, pp.
3253-3264
· 0 citations· 44 references
Medicine
TL;DR
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.
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still a major public health issue, even today. Among the SARS-CoV-2 nonstructural proteins, the main protease (Mpro) plays a critical role in viral polyprotein processing and is therefore indispensable for viral replication. For this reason, it represents one of the most promising therapeutic targets for the development of antiviral agents against SARS-CoV-2. Currently, only one protease antiviral agent (nirmatrelvir) has received emergency approval for COVID-19 treatment, the disease caused by SARS-CoV-2 infection. However, the emergence of viral mutations may compromise its efficacy, highlighting the urgent need to develop new, safe, and effective protease antiviral agents. In the present work, we designed and synthesized new SARS-CoV-2 Mpro small-molecule inhibitors endowed with a pyrimidine scaffold. A series of derivatives were evaluated in both biochemical and cell-based assays to assess their antiviral efficacy, with some of them being able to inhibit the SARS-CoV-2 Mpro activity and to suppress viral replication. Docking studies were confirmed by site-directed mutagenesis, and the mechanism of action of the most promising compound was elucidated.
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 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
Various antiviral drug therapies have been developed for the treatment of coronaviruses, particularly SARS-CoV-2. However, the emergence of drug-resistant mutations is detrimental to clinical efficacy and global public health. Antiviral usage exerts selective pressures on viruses that manufacture an environment for resistant strains to emerge, in some cases at a fitness cost. However, the appearance of a compensatory mutation can restore or improve viral fitness, allowing the strain to persist and spread in a population. Here we evaluate the drug resistance mechanisms of multiple SARS-CoV-2 non-structural proteins, including the main protease (Mpro) and the RNA-dependent RNA polymerase (RdRP), which drive polyprotein processing and viral RNA replication, as well as PLPro, EndoU, and Mac1, which contribute to viral replication and counter host innate immune responses. We also discuss several methods that could be used to avoid drug resistance in the future. By integrating the understanding of molecular mechanisms of antiviral treatment with surveillance of resistance-associated mutations and new drug therapies, appropriate clinical approaches can be developed to reduce the impact of drug resistance.
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.
Quanling Zhang, Tingting Wen, Meng-Si Li et al.· Drug Discoveries & Therapeut...· 0 citations
The SARS-CoV-2 main protease (Mpro) is a key antiviral drug target due to its essential role in the pathogen's replication cycle. The development of potent next-generation Mpro inhibitors is of great importance to ensure the availability of safe and effective COVID-19 therapeutics. Associated research necessitates robust and highly sensitive biochemical assays for the early kinetic characterization of Mpro inhibitor candidates. These assays often reach their limits when characterizing highly potent inhibitors, with a common bottleneck being the insufficient catalytic efficiency and competitive capacity of employed Mpro substrates. To optimize assays for highly active Mpro inhibitors by providing Mpro substrates with improved kinetic features, we created a library of fluorogenic substrates (2-8). These reporters were structurally derived from the well-established Mpro substrate Boc-Abu-Tle-Leu-Gln-AMC (1) and the clinically approved covalent-reversible Mpro inhibitor nirmatrelvir. Kinetic evaluation of 1-8 identified hit compound 6 with about ten-fold improved catalytic efficiency (kcat/Km = 21,400 M-1 s-1) compared to parent substrate 1 (kcat/Km = 2410 M-1 s-1). Substrate 6 was successfully applied for the kinetic characterization of three highly potent Mpro inhibitors. Comparative X-ray crystallographic analyses revealed high similarity in the molecular interactions of the C145A mutant Mpro with nirmatrelvir and with a peptide whose sequence was derived from a natural Mpro substrate. The tetrapeptidic AMC derivative 6 will serve as a valuable biochemical tool contributing to the optimization of SARS-CoV-2 Mpro-directed drug research.
R. Voget, Victoria Steiger, Katharina Sylvester et al.· ACS Chemical Biology· 0 citations
This Account summarizes the group's effort to convert PLpro from a challenging target into a tractable antiviral drug-discovery platform and illustrates how integrated assay development, structural biology, medicinal chemistry, pharmacology, virology, and resistance analysis can transform a challenging viral deubiquitinase into a credible antiviral target.
Jun Wang, Kan Li, Bin Tan· Accounts of Chemical Researc...· 0 citations
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