Proteins are dynamic macromolecules. Knowledge of a protein\'s thermally accessible conformations is critical to determining important transitions and designing therapeutics. Accessible conformations are highly constrained by a protein\'s structure such that concerted structural changes due to external perturbations likely track intrinsic conformational transitions. These transitions can be thought of as paths through a conformational landscape. Crystallographic drug fragment screens are high-throughput perturbation experiments, in which thousands of crystals of a drug target are soaked with small-molecule drug precursors (fragments) and examined for fragment binding, mapping potential drug binding sites on the target protein. Here, we describe an open-source Python package, COLAV (COnformational LAndscape Visualization), to infer conformational landscapes from such large-scale crystallographic perturbation studies. We apply COLAV to drug fragment screens of two medically important systems: protein tyrosine phosphatase 1B (PTP-1B), which regulates insulin signaling, and the SARS CoV-2 Main Protease (MPro). With enough fragment-bound structures, we find that such drug screens also enable detailed mapping of proteins\' conformational landscapes.

Variants of SARS-CoV-2 pose significant challenges in public health due to their increased transmissibility and ability to evade natural immunity, vaccine protection, and monoclonal antibody therapeutics. The emergence of the highly transmissible Omicron variant and subsequent subvariants, characterized by an extensive array of over 32 mutations within the spike protein, intensifies concerns regarding vaccine evasion. In response, multiple antiviral therapeutics have received FDA emergency use approval, targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and main protease (Mpro) regions, known to have relatively fewer mutations across novel variants. In this study, we evaluated the efficacy of nirmatrelvir (PF-07321332) and other clinically significant SARS-CoV-2 antivirals against a diverse panel of SARS-CoV-2 variants, encompassing the newly identified Omicron subvariants XBB1.5 and JN.1, using live-virus antiviral assays. Our findings demonstrate that while the last Omicron subvariants exhibited heightened pathogenicity in our animal model, nirmatrelvir and other clinically relevant antivirals consistently maintained their efficacy against all tested variants, including the XBB1.5 subvariant.

In severe COVID-19 cases, an exacerbated inflammatory response triggers a cytokine storm that can worsen the prognosis. Compounds with both antiviral and anti-inflammatory activities show promise as candidates for COVID-19 therapy, as they potentially act against the SARS-CoV-2 infection regardless of the disease stage. One of the most attractive drug targets among coronaviruses is the main protease (MPro). This enzyme is crucial for cleaving polyproteins into non-structural proteins required for viral replication. The aim of this review was to identify SARS-CoV-2 MPro inhibitors with both antiviral and anti-inflammatory properties. The interactions of the compounds within the SARS-CoV-2 MPro binding site were analyzed through molecular docking when data from crystallographic structures were unavailable. 18 compounds were selected and classified into five different superclasses. Five of them exhibit high potency against MPro: GC-376, baicalein, naringenin, heparin, and carmofur, with IC50 values below 0.2 μM. The MPro inhibitors selected have the potential to alleviate lung edema and decrease cytokine release. These molecules mainly target three critical inflammatory pathways: NF-κB, JAK/STAT, and MAPK, all previously associated with COVID-19 pathogenesis. The structures of the compounds occupy the S1/S2 substrate binding subsite of the MPro. They interact with residues from the catalytic dyad (His41 and Cys145) and/or with the oxyanion hole (Gly143, Ser144, and Cys145), which are pivotal for substrate recognition. The MPro SARS-CoV-2 inhibitors with potential anti-inflammatory activities present here could be optimized for maximum efficacy and safety and be explored as potential treatment of both mild and severe COVID-19.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV) mainly causes acute and severe porcine epidemic diarrhea (PED), and is highly fatal in neonatal piglets. No reliable therapeutics against the infection exist, which poses a major global health issue for piglets. Luteolin is a flavonoid with anti-viral activity toward several viruses.
RESULTS: We evaluated anti-viral effects of luteolin in PEDV-infected Vero and IPEC-J2 cells, and identified IC50 values of 23.87 µM and 68.5 µM, respectively. And found PEDV internalization, replication and release were significantly reduced upon luteolin treatment. As luteolin could bind to human ACE2 and SARS-CoV-2 main protease (Mpro) to contribute viral entry, we first identified that luteolin shares the same core binding site on pACE2 with PEDV-S by molecular docking and exhibited positive pACE2 binding with an affinity constant of 71.6 µM at dose-dependent increases by surface plasmon resonance (SPR) assay. However, pACE2 was incapable of binding to PEDV-S1. Therefore, luteolin inhibited PEDV internalization independent of PEDV-S binding to pACE2. Moreover, luteolin was firmly embedded in the groove of active pocket of Mpro in a three-dimensional docking model, and fluorescence resonance energy transfer (FRET) assays confirmed that luteolin inhibited PEDV Mpro activity. In addition, we also observed PEDV-induced pro-inflammatory cytokine inhibition and Nrf2-induced HO-1 expression. Finally, a drug resistant mutant was isolated after 10 cell culture passages concomitant with increasing luteolin concentrations, with reduced PEDV susceptibility to luteolin identified at passage 10.
CONCLUSIONS: Our results push forward that anti-PEDV mechanisms and resistant-PEDV properties for luteolin, which may be used to combat PED.

Human coronavirus 229E (HCoV-229E) is associated with upper respiratory tract infections and generally causes mild respiratory symptoms. HCoV-229E infection can cause cell death, but the molecular pathways that lead to virus-induced cell death as well as the interplay between viral proteins and cellular cell death effectors remain poorly characterized for HCoV-229E. Studying how HCoV-229E and other common cold coronaviruses interact with and affect cell death pathways may help to understand its pathogenesis and compare it to that of highly pathogenic coronaviruses. Here, we report that the main protease (Mpro) of HCoV-229E can cleave gasdermin D (GSDMD) at two different sites (Q29 and Q193) within its active N-terminal domain to generate fragments that are now unable to cause pyroptosis, a form of lytic cell death normally executed by this protein. Despite GSDMD cleavage by HCoV-229E Mpro, we show that HCoV-229E infection still leads to lytic cell death. We demonstrate that during virus infection caspase-3 cleaves and activates gasdermin E (GSDME), another key executioner of pyroptosis. Accordingly, GSDME knockout cells show a significant decrease in lytic cell death upon virus infection. Finally, we show that HCoV-229E infection leads to increased lytic cell death levels in cells expressing a GSDMD mutant uncleavable by Mpro (GSDMD Q29A+Q193A). We conclude that GSDMD is inactivated by Mpro during HCoV-229E infection, preventing GSDMD-mediated cell death, and point to the caspase-3/GSDME axis as an important player in the execution of virus-induced cell death. In the context of similar reported findings for highly pathogenic coronaviruses, our results suggest that these mechanisms do not contribute to differences in pathogenicity among coronaviruses. Nonetheless, understanding the interactions of common cold-associated coronaviruses and their proteins with the programmed cell death machineries may lead to new clues for coronavirus control strategies.

The coronavirus disease 19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global health crisis with millions of confirmed cases and related deaths. The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication and presents an attractive target for drug development. Despite the approval of some drugs, the search for effective treatments continues. In this study, we systematically evaluated 342 holo-crystal structures of Mpro to identify optimal conformations for structure-based virtual screening (SBVS). Our analysis revealed limited structural flexibility among the structures. Three docking programs, AutoDock Vina, rDock, and Glide were employed to assess the efficiency of virtual screening, revealing diverse performances across selected Mpro structures. We found that the structures 5RHE, 7DDC, and 7DPU (PDB Ids) consistently displayed the lowest EF, AUC, and BEDROCK scores. Furthermore, these structures demonstrated the worst pose prediction results in all docking programs. Two structural differences contribute to variations in docking performance: the absence of the S1 subsite in 7DDC and 7DPU, and the presence of a subpocket in the S2 subsite of 7DDC, 7DPU, and 5RHE. These findings underscore the importance of selecting appropriate Mpro conformations for SBVS, providing valuable insights for advancing drug discovery efforts.

BACKGROUND: The viral main protease (Mpro) of SARS-CoV-2 has been recently proposed as a key target to inhibit virus replication in the host. Therefore, molecules that can bind the catalytic site of Mpro could be considered as potential drug candidates in the treatment of SARS-CoV-2 infections. Here we proposed the application of a state-of-the-art analytical platform which combines metabolomics and protein structure analysis to fish-out potential active compounds deriving from a natural matrix, i.e., a blueberry extract.
METHODS: The experiments focus on finding MS covalent inhibitors of Mpro that contain in their structure a catechol/pyrogallol moiety capable of binding to the nucleophilic amino acids of the enzyme\'s catalytic site.
RESULTS: Among the potential candidates identified, the delphinidin-3-glucoside showed the most promising results. Its antiviral activity has been confirmed in vitro on Vero E6 cells infected with SARS-CoV-2, showing a dose-dependent inhibitory effect almost comparable to the known Mpro inhibitor baicalin. The interaction of delphinidin-3-glucoside with the Mpro pocket observed was also evaluated by computational studies.
CONCLUSIONS: The HRMS analytical platform described proved to be effective in identifying compounds that covalently bind Mpro and are active in the inhibition of SARS-CoV-2 replication, such as delphinidin-3-glucoside.

While research has identified several inhibitors of the main protease (Mpro) of SARS-CoV-2, a significant portion of these compounds exhibit reduced activity in the presence of reducing agents, raising concerns about their effectiveness in vivo. Furthermore, the conventional biosafety level 3 (BSL-3) for cellular assays using viral particles poses a limitation for the widespread evaluation of Mpro inhibitor efficacy in a cell-based assay. Here, we established a BSL-1 compatible cellular assay to evaluate the in vivo potential of Mpro inhibitors. This assay utilizes mammalian cells expressing a tagged Mpro construct containing N-terminal glutathione S-transferase (GST) and C-terminal hemagglutinin (HA) tags and monitors Mpro autodigestion. Using this method, GC376 and boceprevir effectively inhibited Mpro autodigestion, suggesting their potential in vivo activity. Conversely, carmofur and ebselen did not exhibit significant inhibitory effects in this assay. We further investigated the inhibitory potential of selenoneine on Mpro using this approach. Computational analyses of binding energies suggest that noncovalent interactions play a critical role in facilitating the covalent modification of the C145 residue, leading to Mpro inhibition. Our method is straightforward, cost-effective, and readily applicable in standard laboratories, making it accessible to researchers with varying levels of expertise in infectious diseases.

COVID-19 pandemic is predominantly caused by SARS-CoV-2, with its main protease, Mpro, playing a pivotal role in viral replication and serving as a potential target for inhibiting different variants. In this study, potent Mpro inhibitors were identified from glycyrrhizic acid (GL) derivatives with amino acid methyl/ethyl esters. Out of the 17 derivatives semisynthesized, Compounds 2, 6, 9, and 15, with methionine methyl esters, D-tyrosine methyl esters, glutamic acid methyl esters, and methionines in the carbohydrate moiety, respectively, significantly inhibited wild-type SARS-CoV-2 Mpro-mediated proteolysis, with IC50 values ranging from 0.06 μM to 0.84 μM. They also demonstrated efficacy in inhibiting trans-cleavage by mutant Mpro variants (Mpro_P132H, Mpro_E166V, Mpro_P168A, Mpro_Q189I), with IC50 values ranging from 0.05 to 0.92 μM, surpassing nirmatrelvir (IC50: 1.17-152.9 μM). Molecular modeling revealed stronger interactions with Valine166 in the structural complex of Mpro_E166V with the compounds compared to nirmatrelvir. Moreover, these compounds efficiently inhibited the post-entry viral processes of wild-type SARS-CoV-2 single-round infectious particles (SRIPs), mitigating viral cytopathic effects and reducing replicon-driven GFP reporter signals, as well as in vitro infectivity of wild-type, Mpro_E166V, and Mpro_Q189I SRIPs, with EC50 values ranging from 0.02 to 0.53 μM. However, nirmatrelvir showed a significant decrease in inhibiting the replication of mutant SARS-CoV-2 SRIPs carrying Mpro_E166V (EC50: >20 μM) and Mpro_Q189I (EC50: 13.2 μM) compared to wild-type SRIPs (EC50: 0.06 μM). Overall, this study identifies four GL derivatives as promising lead compounds for developing treatments against various SARS-CoV-2 strains, including Omicron, and nirmatrelvir-resistant variants.

Numerous prior studies have identified therapeutic targets that could effectively combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, including the angiotensin-converting enzyme 2 (ACE2) receptor, RNA-dependent RNA polymerase (RdRp), and Main protease (Mpro). In parallel, antiviral compounds like abacavir, acyclovir, adefovir, amantadine, amprenavir, darunavir, didanosine, oseltamivir, penciclovir, and tenofovir are under investigation for their potential in drug repurposing to address this infection. The aim of the study was to determine the effect of modifying the functional groups of the aforementioned antivirals in silico. Using the genetic optimization for ligand docking algorithm on software Maestro (version 11.1), the modified antivirals were docked onto ACE2 receptor, RdRp, and Mpro. Using QuickProp (Maestro v11.1), PASS (prediction of activity spectra for the substances), and altogether with SwissADME, the ADMET (absorption, distribution, metabolism, excretion, and toxicity) of the modified antivirals, as well as their bioavailability and the predicted activity spectra, were determined. Discovery studio software was used to undertake post-docking analysis. Among the 10 antivirals, N(CH3)2 derivative of darunavir, N(CH3)2 derivative of amprenavir and NCH3 derivative of darunavir exhibited best binding affinities with ACE2 receptor (docking scores: -10.333, -9.527 and -9.695 kJ/mol, respectively). Moreover, NCH3 derivative of abacavir (-6.506 kJ/mol), NO2 derivative of didanosine (-6.877 kJ/mol), NCH3 derivative of darunavir (-7.618 kJ/mol) exerted promising affinity to Mpro. In conclusion, the results of the in silico screenings can serve as a useful information for future experimental works.