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The SARS-CoV-2 Main Protease (Mpro), a key enzyme for viral replication, represents a highly attractive target for the development of broad-spectrum anti-coronavirus therapeutics. The organoselenium drug Ebselen has shown potent in vitro inhibition of Mpro as well as antiviral activity, granting clinical interest as a COVID-19 treatment option. Here we show that Ebselen and selected derivatives with human neutrophil elastase (HNE) inhibition and anti-radical activity are able to bind covalently to the viral enzyme with multiple stoichiometry, exhibiting inhibitory activity towards SARS-CoV-2 Mpro with potencies in the nanomolar range. Employing a mass spectrometry-based approach, we show that, upon binding to the target, Ebselen and its derivatives induce a dose-dependent shift in the dimer-monomer equilibrium, favouring the inactive monomeric state of the viral protease and possibly contributing to the observed in vitro inhibition.The SARS-CoV-2 Main Protease (Mpro), a key enzyme for viral replication, represents a highly attractive target for the development of broad-spectrum anti-coronavirus therapeutics. The organoselenium drug Ebselen has shown potent in vitro inhibition of Mpro as well as antiviral activity, granting clinical interest as a COVID-19 treatment option. Here we show that Ebselen and selected derivatives with human neutrophil elastase (HNE) inhibition and anti-radical activity are able to bind covalently to the viral enzyme with multiple stoichiometry, exhibiting inhibitory activity towards SARS-CoV-2 Mpro with potencies in the nanomolar range. Employing a mass spectrometry-based approach, we show that, upon binding to the target, Ebselen and its derivatives induce a dose-dependent shift in the dimer-monomer equilibrium, favouring the inactive monomeric state of the viral protease and possibly contributing to the observed in vitro inhibition.

Recently emerged SARS-CoV-2 caused a major outbreak of coronavirus disease 2019 (COVID-19) and instigated a widespread fear, threatening global health safety. To date, no licensed antiviral drugs or vaccines are available against COVID-19 although several clinical trials are under way to test possible therapies. During this urgent situation, computational drug discovery methods provide an alternative to tiresome high-throughput screening, particularly in the hit-to-lead-optimization stage. Identification of small molecules that specifically target viral replication apparatus has indicated the highest potential towards antiviral drug discovery. In this work, we present potential compounds that specifically target SARS-CoV-2 vital proteins, including the main protease, Nsp12 RNA polymerase and Nsp13 helicase. An integrative virtual screening and molecular dynamics simulations approach has facilitated the identification of potential binding modes and favourable molecular interaction profile of corresponding compounds. Moreover, the identification of structurally important binding site residues in conserved motifs located inside the active site highlights relative importance of ligand binding based on residual energy decomposition analysis. Although the current study lacks experimental validation, the structural information obtained from this computational study has paved way for the design of targeted inhibitors to combat COVID-19 outbreak. [Display omitted] •The integrated structure-based approach identified potential inhibitors against SARS-CoV-2 proteins.•Structurally important binding site residues are elucidated through energy decomposition analysis.•The structural insights provide important information for future drug development against COVID-19.

Nirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration–response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration–response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness.

After almost two years from its first evidence, the COVID-19 pandemic continues to afflict people worldwide, highlighting the need for multiple antiviral strategies. SARS-CoV-2 main protease (Mpro/3CLpro) is a recognized promising target for the development of effective drugs. Because single target inhibition might not be sufficient to block SARS-CoV-2 infection and replication, multi enzymatic-based therapies may provide a better strategy. Here we present a structural and biochemical characterization of the binding mode of MG-132 to both the main protease of SARS-CoV-2, and to the human Cathepsin-L, suggesting thus an interesting scaffold for the development of double-inhibitors. X-ray diffraction data show that MG-132 well fits into the Mpro active site, forming a covalent bond with Cys145 independently from reducing agents and crystallization conditions. Docking of MG-132 into Cathepsin-L well-matches with a covalent binding to the catalytic cysteine. Accordingly, MG-132 inhibits Cathepsin-L with nanomolar potency and reversibly inhibits Mpro with micromolar potency, but with a prolonged residency time. We compared the apo and MG-132-inhibited structures of Mpro solved in different space groups and we identified a new apo structure that features several similarities with the inhibited ones, offering interesting perspectives for future drug design and in silico efforts.

With the continual evolution of new strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The SARS-CoV-2 main protease (M pro ) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting M pro appear promising but will elicit selection pressure for resistance. To understand resistance potential in M pro , we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high throughput assays of M pro function in yeast, based on either the ability of M pro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to M pro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation, making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the M pro dimer. The clinical variants of M pro were predominantly functional in our screens, indicating that M pro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to M pro evolution and that are likely to contribute to drug resistance. This complete mutational guide of M pro can be used in the design of inhibitors with reduced potential of evolving viral resistance.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to represent a global health emergency as a highly transmissible, airborne virus. An important coronaviral drug target for treatment of COVID-19 is the conserved main protease (Mpro). Nirmatrelvir is a potent Mpro inhibitor and the antiviral component of Paxlovid. The significant viral sequencing effort during the ongoing COVID-19 pandemic represented a unique opportunity to assess potential nirmatrelvir escape mutations from emerging variants of SARS-CoV-2. To establish the baseline mutational landscape of Mpro prior to the introduction of Mpro inhibitors, Mpro sequences and its cleavage junction regions were retrieved from ~4,892,000 high-quality SARS-CoV-2 genomes in the open-access Global Initiative on Sharing Avian Influenza Data (GISAID) database. Any mutations identified from comparison to the reference sequence (Wuhan-Hu-1) were catalogued and analyzed. Mutations at sites key to nirmatrelvir binding and protease functionality (e.g., dimerization sites) were still rare. Structural comparison of Mpro also showed conservation of key nirmatrelvir contact residues across the extended Coronaviridae family (α-, β-, and γ-coronaviruses). Additionally, we showed that over time, the SARS-CoV-2 Mpro enzyme remained under purifying selection and was highly conserved relative to the spike protein. Now, with the emergency use authorization (EUA) of Paxlovid and its expected widespread use across the globe, it is essential to continue large-scale genomic surveillance of SARS-CoV-2 Mpro evolution. This study establishes a robust analysis framework for monitoring emergent mutations in millions of virus isolates, with the goal of identifying potential resistance to present and future SARS-CoV-2 antivirals. IMPORTANCE The recent authorization of oral severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antivirals, such as Paxlovid, has ushered in a new era of the COVID-19 pandemic. The emergence of new variants, as well as the selective pressure imposed by antiviral drugs themselves, raises concern for potential escape mutations in key drug binding motifs. To determine the potential emergence of antiviral resistance in globally circulating isolates and its implications for the clinical response to the COVID-19 pandemic, sequencing of SARS-CoV-2 viral isolates before, during, and after the introduction of new antiviral treatments is critical. The infrastructure built herein for active genetic surveillance of Mpro evolution and emergent mutations will play an important role in assessing potential antiviral resistance as the pandemic progresses and Mpro inhibitors are introduced. We anticipate our framework to be the starting point in a larger effort for global monitoring of the SARS-CoV-2 Mpro mutational landscape.

Since the outbreak of COVID-19, one of the strategies used to search for new drugs has been to find inhibitors of the main protease (Mpro) of the virus SARS-CoV-2. Initially, previously reported inhibitors of related proteases such as the main proteases of SARS-CoV and MERS-CoV were tested. A huge effort was then carried out by the scientific community to design, synthesize and test new small molecules acting as inactivators of SARS-CoV-2 Mpro. From the chemical structure view, these compounds can be classified into two main groups: one corresponds to modified peptides displaying an adequate sequence for high affinity and a reactive warhead; and the second is a diverse group including chemical compounds that do not have a peptide framework. Although a drug including a SARS-CoV-2 main protease inhibitor has already been commercialized, denoting the importance of this field, more compounds have been demonstrated to be promising potent inhibitors as potential antiviral drugs.

The uncontrolled spread of the COVID-19 pandemic caused by the new coronavirus SARS-CoV-2 during 2020–2021 is one of the most devastating events in the history, with remarkable impacts on the health, economic systems, and habits of the entire world population. While some effective vaccines are nowadays approved and extensively administered, the long-term efficacy and safety of this line of intervention is constantly under debate as coronaviruses rapidly mutate and several SARS-CoV-2 variants have been already identified worldwide. Then, the WHO’s main recommendations to prevent severe clinical complications by COVID-19 are still essentially based on social distancing and limitation of human interactions, therefore the identification of new target-based drugs became a priority. Several strategies have been proposed to counteract such viral infection, including the repurposing of FDA already approved for the treatment of HIV, HCV, and EBOLA, inter alia. Among the evaluated compounds, inhibitors of the main protease of the coronavirus (Mpro) are becoming more and more promising candidates. Mpro holds a pivotal role during the onset of the infection and its function is intimately related with the beginning of viral replication. The interruption of its catalytic activity could represent a relevant strategy for the development of anti-coronavirus drugs. SARS-CoV-2 Mpro is a peculiar cysteine protease of the coronavirus family, responsible for the replication and infectivity of the parasite. This review offers a detailed analysis of the repurposed drugs and the newly synthesized molecules developed to date for the treatment of COVID-19 which share the common feature of targeting SARS-CoV-2 Mpro, as well as a brief overview of the main enzymatic and cell-based assays to efficaciously screen such compounds.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been validated as a therapeutic target for antiviral drug development, given its critical role in the viral life cycle. SARS-CoV-2 Mpro contains 12 cysteine residues, which are susceptible to covalent modification by nucleophilic entities. In this study, we showcase an efficient strategy to uncover the key covalent inhibitors of SARS-CoV-2 Mpro from herbal extracts and decipher their synergistic anti-Mpro mechanisms. Preliminary screening identified Rhodiola crenulata root (RCR), a well-known Tibetan herb, showing the most potent time-dependent inhibition against SARS-CoV-2 Mpro. By integrating fluorescence resonance energy transfer (FRET)-based biochemical assay with phytochemical and chemoproteomic profiling, we efficiently identified thirteen Mpro covalent inhibitors from the crude extract of RCR. Among these, rhodiosin and gallic acid were validated as the key anti-Mpro constituents, due to their strong anti-Mpro effects and high abundance in RCR. Remarkably, their combination exhibited a pronounced synergy in Mpro inhibition. Further intact protein mass measurements and top-down mass spectrometry (MS) analysis, complemented by biophysical methods, elucidated how these two compounds work in concert. Our findings revealed that rhodiosin functions as an allosteric inhibitor, disrupting Mpro dimerization and significantly facilitating the covalent modification of Mpro by gallic acid. Collectively, the covalent SARS-CoV-2 Mpro inhibitors are efficiently identified from a Tibetan herb, while a phytochemical combination with synergistic anti-Mpro effects and their unique allosteric-induced cooperative modification mechanism are revealed. [Display omitted] •RCR showed potent anti-SARS-CoV-2 Mpro activity.•An integrated strategy was employed to discover the covalent inhibitors of Mpro in RCR.•A total of 13 compounds in RCR were found to covalently modify the cysteines of Mpro.•Rho and GA drug-pair inhibit Mpro via allosteric-induced cooperative modification.•TD-MS uncover the key modified sites and the synergistic mechanism of Rho and GA.

The coronavirus disease 2019 (COVID-19) has spread widely around the world and has seriously affected the human health of tens of millions of people. In view of lacking anti-virus drugs target to SARS-CoV-2, there is an urgent need to develop effective new drugs. In this study, we reported our discovery of SARS-CoV-2 M pro inhibitors. We selected 15 natural compounds, including 7 flavonoids, 3 coumarins, 2 terpenoids, one henolic, one aldehyde and one steroid compound for molecular docking and enzymatic screening. Myricetin were identified to have potent inhibit activity with IC 50 3.684 ± 0.076 μM in the enzyme assay. The binding pose of Myricetin with SARS-CoV-2 M pro was identified using molecular docking method. In the binding pocket of SARS-CoV-2 M pro , the chromone ring of Myricetin interacts with His41 through π -π stacking, and the 3’-, 4’- and 7-hydroxyl of Myricetin interact with Phe140, Glu166and Asp187 through hydrogen bonds. Significantly, our results showed that Myricetin has potent effect on bleomycin-induced pulmonary inflammation by inhibiting the infiltration of inflammatory cells and the secretion of inflammatory cytokines IL-6, IL-1α, TNF-α and IFN-γ. Overall, Myricetin may be a potential drug for anti-virus and symptomatic treatment of COVID-19.