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Human enteroviruses are important pathogens of hand-foot-and-mouth disease, poliomyelitis, and encephalitis, etc., posing substantial global health burdens with no specific approved therapeutics. While traditional Chinese medicine (TCM) has demonstrated antiviral potential during the COVID-19 pandemic, its efficacy and pharmacodynamic material basis against enteroviruses remains underexplored. Here, we systematically characterized the broad-spectrum anti-enterovirus activity of Huashi Baidu Formula (HSBDF), a clinically approved TCM for COVID-19, and identified three flavonoid compounds as its active components responsible for this antiviral effect. Transcriptomics analysis revealed that HSBDF attenuated CV-A9-induced inflammation by modulating MAPK and NF-κB signaling pathways. High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) analysis identified 152 chemical compounds in HSBDF, among which three flavonoids—velutin, isorhamnetin, and (−)-epicatechin gallate—exhibited potent pan-enteroviral inhibition. Mechanistically, these compounds suppressed the activity of 3C proteases in enteroviruses, while concurrently attenuating CV-A9-induced upregulation of IL-6, TNF-α, MCP-1, and COX-2. Utilizing a BALB/c young mouse model, it was demonstrated that the HSBDF and its compound velutin effectively suppressed viral replication in vivo. Collectively, this study advances TCM-based strategies for enterovirus therapy exemplified by HSBDF and highlights flavonoid scaffolds as promising candidates for developing broad-spectrum anti-enteroviral agents.

Severe Acute Respiratory Syndrome caused by a coronavirus is a recent viral infection. There is no scientific evidence or clinical trials to indicate that possible therapies have demonstrated results in suspected or confirmed patients. This work aims to perform a virtual screening of 1430 ligands through molecular docking and to evaluate the possible inhibitory capacity of these drugs about the Mpro protease of Covid-19. The selected drugs were registered with the FDA and available in the virtual drug library, widely used by the population. The simulation was performed using the MolAiCalD algorithm, with a Lamarckian genetic model (GA) combined with energy estimation based on rigid and flexible conformation grids. In addition, molecular dynamics studies were also performed to verify the stability of the receptor-ligand complexes formed through analyses of RMSD, RMSF, H–Bond, SASA, and MMGBSA. Compared to the binding energy of the synthetic redocking coupling (−6.8 kcal/mol/RMSD of 1.34 Å), which was considerably higher, it was then decided to analyze the parameters of only three ligands: ergotamine (−9.9 kcal/mol/RMSD of 2.0 Å), dihydroergotamine (−9.8 kcal/mol/RMSD of 1.46 Å) and olysio (−9.5 kcal/mol/RMSD of 1.5 Å). It can be stated that ergotamine showed the best interactions with the Mpro protease of Covid-19 in the in silico study, showing itself as a promising candidate for treating Covid-19.

Nirmatrelvir is a specific antiviral drug that targets the main protease (M pro ) of SARS-CoV-2 and has been approved to treat COVID-19 1 , 2 . As an RNA virus characterized by high mutation rates, whether SARS-CoV-2 will develop resistance to nirmatrelvir is a question of concern. Our previous studies have shown that several mutational pathways confer resistance to nirmatrelvir, but some result in a loss of viral replicative fitness, which is then compensated for by additional alterations 3 . The molecular mechanisms for this observed resistance are unknown. Here we combined biochemical and structural methods to demonstrate that alterations at the substrate-binding pocket of M pro can allow SARS-CoV-2 to develop resistance to nirmatrelvir in two distinct ways. Comprehensive studies of the structures of 14 M pro mutants in complex with drugs or substrate revealed that alterations at the S1 and S4 subsites substantially decreased the level of inhibitor binding, whereas alterations at the S2 and S4′ subsites unexpectedly increased protease activity. Both mechanisms contributed to nirmatrelvir resistance, with the latter compensating for the loss in enzymatic activity of the former, which in turn accounted for the restoration of viral replicative fitness, as observed previously 3 . Such a profile was also observed for ensitrelvir, another clinically relevant M pro inhibitor. These results shed light on the mechanisms by which SARS-CoV-2 evolves to develop resistance to the current generation of protease inhibitors and provide the basis for the design of next-generation M pro inhibitors. A biochemical and structural analysis demonstrates that alterations at the substrate-binding pocket of the main protease of SARS-CoV-2 can allow the virus to develop resistance to nirmatrelvir in two distinct ways.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2–treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

Human noroviruses (HuNoVs) are the primary cause of acute viral gastroenteritis. There are no antivirals or vaccines available to treat and/or prevent HuNoV. Norovirus 3C-like protease (3CLpro) is essential for viral replication; consequently, the inhibition of this enzyme is a fruitful avenue for antinorovirus therapeutics. To discover novel 3CLpro inhibitors with diverse scaffolds, a multi-stage virtual screening approach was performed by docking >10 million compounds into the 3CLpro catalytic site. An initial subset of 18 compounds was selected, and compounds YY-1029 and YY-4204 were identified as the best two molecules. Molecular dynamics (MD) simulations and binding free energy calculations (MM/GBSA) of YY-1029 and YY-4204 were performed to elucidate the binding mechanisms. The ADMET properties were also estimated to predict the potential druggability of representative molecules. All 18 compounds were evaluated for their antinorovirus activity and cytotoxicity in a cell-based replicon system. This work could provide information for the development of 3CL pro inhibitors.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment. The 3CL protease of SARS-CoV-2 is inhibited by PF-00835231 in vitro. Here, the authors show that the prodrug PF-07304814 has broad spectrum activity, inhibiting SARS-CoV and SARS-CoV-2 in mice and its ADME and safety profile support clinical development.

Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (Mpro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 Mpro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 Mpro at 2.5 µM, which is more potent than against SAR-CoV-1 Mpro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 Mpro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19.

The SARS-CoV-2 3CL protease is a critical drug target for small molecule COVID-19 therapy, given its likely druggability and essentiality in the viral maturation and replication cycle. Based on the conservation of 3CL protease substrate binding pockets across coronaviruses and using screening, we identified four structurally distinct lead compounds that inhibit SARS-CoV-2 3CL protease. After evaluation of their binding specificity, cellular antiviral potency, metabolic stability, and water solubility, we prioritized the GC376 scaffold as being optimal for optimization. We identified multiple drug-like compounds with <10 nM potency for inhibiting SARS-CoV-2 3CL and the ability to block SARS-CoV-2 replication in human cells, obtained co-crystal structures of the 3CL protease in complex with these compounds, and determined that they have pan-coronavirus activity. We selected one compound, termed coronastat, as an optimized lead and characterized it in pharmacokinetic and safety studies in vivo. Coronastat represents a new candidate for a small molecule protease inhibitor for the treatment of SARS-CoV-2 infection for eliminating pandemics involving coronaviruses. Small molecule drugs promise to remain a valuable tool in controlling the ongoing COVID-19 pandemic. Here the authors describe optimized drug-like small molecule inhibitors of the SARS-CoV-2 3CL protease for potential treatment of COVID-19.

The Coronavirus disease 2019 (COVID-19), caused by the novel coronavirus, SARS-CoV-2, has recently emerged as a pandemic. Here, an attempt has been made through in-silico high throughput screening to explore the antiviral compounds from traditionally used plants for antiviral treatments in India namely, Tea, Neem and Turmeric, as potential inhibitors of two widely studied viral proteases, main protease (Mpro) and papain-like protease (PLpro) of the SARS-CoV-2. Molecular docking study using BIOVIA Discovery Studio 2018 revealed, (−)-epicatechin-3-O-gallate (ECG), a tea polyphenol has a binding affinity toward both the selected receptors, with the lowest CDocker energy − 46.22 kcal mol−1 for SARS-CoV-2 Mpro and CDocker energy − 44.72 kcal mol−1 for SARS-CoV-2 PLpro, respectively. The SARS-CoV-2 Mpro complexed with (−)-epicatechin-3-O-gallate, which had shown the best binding affinity was subjected to molecular dynamics simulations to validate its binding affinity, during which, the root-mean-square-deviation values of SARS-CoV-2 Mpro–Co-crystal ligand (N3) and SARS-CoV-2 Mpro- (−)-epicatechin-3-O-gallate systems were found to be more stable than SARS-CoV-2 Mpro system. Further, (−)-epicatechin-3-O-gallate was subjected to QSAR analysis which predicted IC50 of 0.3281 nM against SARS-CoV-2 Mpro. Overall, (−)-epicatechin-3-O-gallate showed a potential binding affinity with SARS-CoV-2 Mpro and could be proposed as a potential natural compound for COVID-19 treatment.Graphic abstract

The RNA-dependent RNA polymerase (RdRp), 3C-like protease (3CL pro ), and papain-like protease (PL pro ) are pivotal components in the viral life cycle of SARS-CoV-2, presenting as promising therapeutic targets. Currently, all FDA-approved antiviral drugs against SARS-CoV-2 are RdRp or 3CL pro inhibitors. However, the mutations causing drug resistance have been observed in RdRp and 3CL pro from SARS-CoV-2, which makes it necessary to develop antivirals with novel mechanisms. Through the application of a structure-based drug design (SBDD) approach, we discover a series of novel potent non-covalent PL pro inhibitors with remarkable in vitro potency and in vivo PK properties. The co-crystal structures of PL pro with lead compounds reveal that the residues D164 and Q269 around the S2 site are critical for improving the inhibitor’s potency. The lead compound GZNL-P36 not only inhibits SARS-CoV-2 and its variants at the cellular level with EC 50 ranging from 58.2 nM to 306.2 nM, but also inhibits HCoV-NL63 and HCoV-229E with EC 50 of 81.6 nM and 2.66 μM, respectively. Oral administration of the GZNL-P36 results in significantly improved survival and notable reductions in lung viral loads and lesions in SARS-CoV-2 infection mouse model, consistent with RNA-seq data analysis. Our results indicate that PL pro inhibitors represent a promising SARS-CoV-2 therapy. In this work, the authors present novel PLpro inhibitors, with lead compound GZNL-P36 showing potent activity against SARS-CoV-2 variants, improving survival and reducing lung viral loads in a mouse model, offering promise for COVID−19 therapies.