Host Cell Proteases Involved in Human Respiratory Viral Infections and Their Inhibitors: A Review.

Viral tropism is most commonly linked to receptor use, but host cell protease use can be a notable factor in susceptibility to infection. Here we review the use of host cell proteases by human viruses, focusing on those with primarily respiratory tropism, particularly SARS-CoV-2. We first describe the various classes of proteases present in the respiratory tract, as well as elsewhere in the body, and incorporate the targeting of these proteases as therapeutic drugs for use in humans. Host cell proteases are also linked to the systemic spread of viruses and play important roles outside of the respiratory tract; therefore, we address how proteases affect viruses across the spectrum of infections that can occur in humans, intending to understand the extrapulmonary spread of SARS-CoV-2.

Development of a Biosafety Level 1 Cellular Assay for Identifying Small-Molecule Antivirals Targeting the Main Protease of SARS-CoV-2: Evaluation of Cellular Activity of GC376, Boceprevir, Carmofur, Ebselen, and Selenoneine.

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.

Shedding Light on Dark Chemical Matter: The Discovery of a SARS-CoV-2 Mpro Main Protease Inhibitor through Intensive Virtual Screening and In Vitro Evaluation.

The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.

[A Bioinformatics Method for Identification of Human Proteases Active against Viral Envelope Glycoproteins: A Case Study on the SARS-CoV-2 Spike Protein].

Many viruses, including SARS-CoV-2, the coronavirus responsible for the COVID-19 pandemic, enter host cells through a process of cell-viral membrane fusion that is activated by proteolytic enzymes. Typically, these enzymes are host cell proteases. Identifying the proteases that activate the virus is not a simple task but is important for the development of new antiviral drugs. In this study, we developed a bioinformatics method for identifying proteases that can cleave viral envelope glycoproteins. The proposed approach involves the use of predictive models for the substrate specificity of human proteases and the application of a structural analysis method for predicting the vulnerability of protein regions to proteolysis based on their 3D structures. Specificity models were constructed for 169 human proteases using information on their known substrates. A previously developed method for structural analysis of potential proteolysis sites was applied in parallel with specificity models. Validation of the proposed approach was performed on the SARS-CoV-2 spike protein, whose proteolysis sites have been well studied.

Structure-based virtual screening methods for the identification of novel phytochemical inhibitors targeting furin protease for the management of COVID-19.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is a highly contagious respiratory disease with widespread societal impact. The symptoms range from cough, fever, and pneumonia to complications affecting various organs, including the heart, kidneys, and nervous system. Despite various ongoing efforts, no effective drug has been developed to stop the spread of the virus. Although various types of medications used to treat bacterial and viral diseases have previously been employed to treat COVID-19 patients, their side effects have also been observed. The way SARS-CoV-2 infects the human body is very specific, as its spike protein plays an important role. The S subunit of virus spike protein cleaved by human proteases, such as furin protein, is an initial and important step for its internalization into a human host. Keeping this context, we attempted to inhibit the furin using phytochemicals that could produce minimal side effects. For this, we screened 408 natural phytochemicals from various plants having antiviral properties, against furin protein, and molecular docking and dynamics simulations were performed. Based on the binding score, the top three compounds (robustaflavone, withanolide, and amentoflavone) were selected for further validation. MM/GBSA energy calculations revealed that withanolide has the lowest binding energy of -57.2 kcal/mol followed by robustaflavone and amentoflavone with a binding energy of -45.2 kcal/mol and -39.68 kcal/mol, respectively. Additionally, ADME analysis showed drug-like properties for these three lead compounds. Hence, these natural compounds robustaflavone, withanolide, and amentoflavone, may have therapeutic potential for the management of SARS-CoV-2 by targeting furin.

3-Chymotrypsin-like Protease (3CLpro) of SARS-CoV-2: Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials.

Proteases represent common targets in combating infectious diseases, including COVID-19. The 3-chymotrypsin-like protease (3CLpro) is a validated molecular target for COVID-19, and it is key for developing potent and selective inhibitors for inhibiting viral replication of SARS-CoV-2. In this review, we discuss structural relationships and diverse subsites of 3CLpro, shedding light on the pivotal role of dimerization and active site architecture in substrate recognition and catalysis. Our analysis of bioinformatics and other published studies motivated us to investigate a novel catalytic mechanism for the SARS-CoV-2 polyprotein cleavage by 3CLpro, centering on the triad mechanism involving His41-Cys145-Asp187 and its indispensable role in viral replication. Our hypothesis is that Asp187 may participate in modulating the pKa of the His41, in which catalytic histidine may act as an acid and/or a base in the catalytic mechanism. Recognizing Asp187 as a crucial component in the catalytic process underscores its significance as a fundamental pharmacophoric element in drug design. Next, we provide an overview of both covalent and non-covalent inhibitors, elucidating advancements in drug development observed in preclinical and clinical trials. By highlighting various chemical classes and their pharmacokinetic profiles, our review aims to guide future research directions toward the development of highly selective inhibitors, underscore the significance of 3CLpro as a validated therapeutic target, and propel the progression of drug candidates through preclinical and clinical phases.

Exploring Cannabinoids as Potential Inhibitors of SARS-CoV-2 Papain-like Protease: Insights from Computational Analysis and Molecular Dynamics Simulations.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global COVID-19 pandemic, challenging healthcare systems worldwide. Effective therapeutic strategies against this novel coronavirus remain limited, underscoring the urgent need for innovative approaches. The present research investigates the potential of cannabis compounds as therapeutic agents against SARS-CoV-2 through their interaction with the virus's papain-like protease (PLpro) protein, a crucial element in viral replication and immune evasion. Computational methods, including molecular docking and molecular dynamics (MD) simulations, were employed to screen cannabis compounds against PLpro and analyze their binding mechanisms and interaction patterns. The results showed cannabinoids with binding affinities ranging from -6.1 kcal/mol to -4.6 kcal/mol, forming interactions with PLpro. Notably, Cannabigerolic and Cannabidiolic acids exhibited strong binding contacts with critical residues in PLpro's active region, indicating their potential as viral replication inhibitors. MD simulations revealed the dynamic behavior of cannabinoid-PLpro complexes, highlighting stable binding conformations and conformational changes over time. These findings shed light on the mechanisms underlying cannabis interaction with SARS-CoV-2 PLpro, aiding in the rational design of antiviral therapies. Future research will focus on experimental validation, optimizing binding affinity and selectivity, and preclinical assessments to develop effective treatments against COVID-19.

In Vitro and in Silico Study of New Biscoumarin Glycosides from Paramignya trimera against Angiotensin-Converting Enzyme 2 (ACE-2) for Preventing SARS-CoV-2 Infection.

In Vietnam, the stems and roots of the Rutaceous plant Paramignya trimera (Oliv.) Burkill (known locally as "Xáo tam phân") are widely used to treat liver diseases such as viral hepatitis and acute and chronic cirrhosis. In an effort to search for Vietnamese natural compounds capable of inhibiting coronavirus based on molecular docking screening, two new dimeric coumarin glycosides, namely cis-paratrimerin B (1) and cis-paratrimerin A (2), and two previously identified coumarins, the trans-isomers paratrimerin B (3) and paratrimerin A (4), were isolated from the roots of P. trimera and tested for their anti-angiotensin-converting enzyme 2 (ACE-2) inhibitory properties in vitro. It was discovered that ACE-2 enzyme was inhibited by cis-paratrimerin B (1), cis-paratrimerin A (2), and trans-paratrimerin B (3), with IC50 values of 28.9, 68, and 77 µM, respectively. Docking simulations revealed that four biscoumarin glycosides had good binding energies (∆G values ranging from -10.6 to -14.7 kcal/mol) and mostly bound to the S1' subsite of the ACE-2 protein. The key interactions of these natural ligands include metal chelation with zinc ions and multiple H-bonds with Ser128, Glu145, His345, Lys363, Thr371, Glu406, and Tyr803. Our findings demonstrated that biscoumarin glycosides from P. trimera roots occur naturally in both cis- and trans-diastereomeric forms. The biscoumarin glycosides Lys363, Thr371, Glu406, and Tyr803. Our findings demonstrated that biscoumarin glycosides from P. trimera roots hold potential for further studies as natural ACE-2 inhibitors for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Structural basis of rosmarinic acid inhibitory mechanism on SARS-CoV-2 main protease.

The SARS-CoV-2 coronavirus is characterized by high mutation rates and significant infectivity, posing ongoing challenges for therapeutic intervention. To address potential challenges in the future, the continued development of effective drugs targeting SARS-CoV-2 remains an important task for the scientific as well as the pharmaceutical community. The main protease (Mpro) of SARS-CoV-2 is an ideal therapeutic target for COVID-19 drug development, leading to the introduction of various inhibitors, both covalent and non-covalent, each characterized by unique mechanisms of action and possessing inherent strengths and limitations. Natural products, being compounds naturally present in the environment, offer advantages such as low toxicity and diverse activities, presenting a viable source for antiviral drug development. Here, we identified a natural compound, rosmarinic acid, which exhibits significant inhibitory effects on the Mpro of the SARS-CoV-2. Through detailed structural biology analysis, we elucidated the precise crystal structure of the complex formed between rosmarinic acid and SARS-CoV-2 Mpro, revealing the molecular basis of its inhibitory mechanism. These findings not only enhance our understanding of the antiviral action of rosmarinic acid, but also provide valuable structural information and mechanistic insights for the further development of therapeutic strategies against SARS-CoV-2.

Generalized open-source workflows for atomistic molecular dynamics simulations of viral helicases.

Viral helicases are promising targets for the development of antiviral therapies. Given their vital function of unwinding double-stranded nucleic acids, inhibiting them blocks the viral replication cycle. Previous studies have elucidated key structural details of these helicases, including the location of substrate binding sites, flexible domains, and the discovery of potential inhibitors. Here we present a series of new Galaxy tools and workflows for performing and analyzing molecular dynamics simulations of viral helicases. We first validate them by demonstrating recapitulation of data from previous simulations of Zika (NS3) and SARS-CoV-2 (NSP13) helicases in apo and complex with inhibitors. We further demonstrate the utility and generalizability of these Galaxy workflows by applying them to new cases, proving their usefulness as a widely accessible method for exploring antiviral activity.

Exploring the Therapeutic Potential of Petiveria alliacea L. Phytochemicals: A Computational Study on Inhibiting SARS-CoV-2's Main Protease (Mpro).

The outbreak of SARS-CoV-2, also known as the COVID-19 pandemic, is still a critical risk factor for both human life and the global economy. Although, several promising therapies have been introduced in the literature to inhibit SARS-CoV-2, most of them are synthetic drugs that may have some adverse effects on the human body. Therefore, the main objective of this study was to carry out an in-silico investigation into the medicinal properties of Petiveria alliacea L. (P. alliacea L.)-mediated phytocompounds for the treatment of SARS-CoV-2 infections since phytochemicals have fewer adverse effects compared to synthetic drugs. To explore potential phytocompounds from P. alliacea L. as candidate drug molecules, we selected the infection-causing main protease (Mpro) of SARS-CoV-2 as the receptor protein. The molecular docking analysis of these receptor proteins with the different phytocompounds of P. alliacea L. was performed using AutoDock Vina. Then, we selected the three top-ranked phytocompounds (myricitrin, engeletin, and astilbin) as the candidate drug molecules based on their highest binding affinity scores of -8.9, -8.7 and -8.3 (Kcal/mol), respectively. Then, a 100 ns molecular dynamics (MD) simulation study was performed for their complexes with Mpro using YASARA software, computed RMSD, RMSF, PCA, DCCM, MM/PBSA, and free energy landscape (FEL), and found their almost stable binding performance. In addition, biological activity, ADME/T, DFT, and drug-likeness analyses exhibited the suitable pharmacokinetics properties of the selected phytocompounds. Therefore, the results of this study might be a useful resource for formulating a safe treatment plan for SARS-CoV-2 infections after experimental validation in wet-lab and clinical trials.

Screening of Mpro Protease (SARS-CoV-2) Covalent Inhibitors from an Anthocyanin-Rich Blueberry Extract Using an HRMS-Based Analytical Platform.

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.

Structural basis for the inhibition of the HCoV-NL63 main protease Mpro by X77.

Human coronaviruses are a group of pathogens that primarily cause respiratory and intestinal diseases. Infection can easily cause respiratory symptoms, as well as a variety of serious complications. There are several types of human coronaviruses, such as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, and SARS-CoV-2. The prevalence of COVID-19 has led to a growing focus on drug research against human coronaviruses. The main protease (Mpro) from human coronaviruses is a relatively conserved that controls viral replication. X77 was discovered to have extremely high inhibitory activity against SARS-CoV-2 Mpro through the use of computer-simulated docking. In this paper, we have resolved the crystal structure of the HCoV-NL63 Mpro complexed with X77 and analyzed their interaction in detail. This data provides essential information for solving their binding modes and their structural determinants. Then, we compared the binding modes of X77 with SARS-CoV-2 Mpro and HCoV-NL63 Mpro in detail. This study illustrates the structural basis of HCoV-NL63 Mpro binding to the inhibitor X77. The structural insights derived from this study will inform the development of new drugs with broad-spectrum resistance to human coronaviruses.

Computational evaluation and benchmark study of 342 crystallographic holo-structures of SARS-CoV-2 Mpro enzyme.

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.

The Identification of Peptide Inhibitors of the Coronavirus 3CL Protease from a Fucus ceranoides L. Hydroalcoholic Extract Using a Ligand-Fishing Strategy.

Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme's proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease's active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.

Deciphering inhibitory activity of marine algae Ecklonia cava phlorotannins against SARS CoV-2 main protease: A coupled in-silico docking and molecular dynamics simulation study.

The onset of COVID-19 due to the SARS CoV-2 virus has spurred an urgent need for potent therapeutics and vaccines to combat this global pandemic. The main protease (Mpro) of the virus, crucial in its replication, has become a focal point in developing anti-COVID-19 drugs. The cysteine protease Mpro in SARS CoV-2 bears a significant resemblance to the same protease found in SARS CoV-1. Previous research highlighted phlorotannins derived from Ecklonia cava, an edible marine algae, as inhibitors of SARS CoV-1 Mpro activity. However, it remains unclear whether these marine-derived phlorotannins also exert a similar inhibitory effect on SARS CoV-2 Mpro. To unravel this, our study utilized diverse in-silico methodologies. We explored the pharmacological potential of various phlorotannins (phloroglucinol, triphloretol-A, eckol, 2-phloroeckol, 7-phloroeckol, fucodiphloroethol G, dieckol, and phlorofucofuroeckol-A) and assessed their binding efficacies alongside established Mpro inhibitors (N3 and lopinavir) through molecular docking studies. Among these compounds, five phlorotannins (eckol, 2-phloroeckol, 7-phloroeckol, dieckol, and phlorofucofuroeckol-A) exhibited potent binding affinities comparable to or surpassing N3 and lopinavir, interacting especially with the catalytic residues His41 and Cys145 of Mpro. Moreover, molecular dynamics simulations revealed that these five Mpro-phlorotannin complexes displayed enhanced stability and maintained comparable or slightly reduced compactness. They exhibited reduced conformational changes and increased expansion relative to the Mpro-N3 and/or Mpro-lopinavir complex. Our MM-GBSA analysis further supported these findings. Overall, our investigation highlights the potential of these five phlorotannins in inhibiting the proteolytic function of SARS CoV-2 Mpro, offering promise for anti-COVID-19 drug development.

Optimization of quenched fluorescent peptide substrates of SARS-CoV-2 3CLpro main protease (Mpro) from proteomic identification of P6-P6' active site specificity.

SARS-CoV-2 3C-like main protease (3CLpro) is essential for protein excision from the viral polyprotein. 3CLpro inhibitor drug development to block SARS-CoV-2 replication focuses on the catalytic non-prime (P) side for specificity and potency, but the importance of the prime (P') side in substrate specificity and for drug development remains underappreciated. We determined the P6-P6' specificity for 3CLpro from >800 cleavage sites that we identified using Proteomic Identification of Cleavage site Specificity (PICS). Cleavage occurred after the canonical P1-Gln and non-canonical P1-His and P1-Met residues. Moreover, P3 showed a preference for Arg/Lys and P3' for His. Essential H-bonds between the N-terminal Ser1 of protomer-B in 3CLpro dimers form with P1-His, but not with P1-Met. Nonetheless, cleavage occurs at P1-Met456 in native MAP4K5. Elevated reactive oxygen species in SARS-CoV-2 infection oxidize methionines. Molecular simulations revealed P1-MetOX forms an H-bond with Ser1 and notably, strong positive cooperativity between P1-Met with P3'-His was revealed, which enhanced peptide-cleavage rates. The highly plastic S3' subsite accommodates P3'-His that displays stabilizing backbone H-bonds with Thr25 lying central in a "'threonine trio" (Thr24-Thr25-Thr26) in the P'-binding domain I. Molecular docking simulations unveiled structure-activity relationships impacting 3CLpro-substrate interactions, and the role of these structural determinants was confirmed by MALDI-TOF-MS cleavage assays of P1'- and P3'-positional scanning peptide libraries carrying a 2nd optimal cut-site as an internal positive control. These data informed the design of two new and highly soluble 3CLproquenched-fluorescent peptide substrates for improved FRET monitoring of 3CLpro activity with 15× improved sensitivity over current assays.IMPORTANCEFrom global proteomics identification of >800 cleavage sites, we characterized the P6-P6' active site specificity of SARS-CoV-2 3CLpro using proteome-derived peptide library screens, molecular modeling simulations, and focussed positional peptide libraries. In P1', we show that alanine and serine are cleaved 3× faster than glycine and the hydrophobic small amino acids Leu, Ile, or Val prevent cleavage of otherwise optimal non-prime sequences. In characterizing non-canonical non-prime P1 specificity, we explored the unusual P1-Met specificity, discovering enhanced cleavage when in the oxidized state (P1-MetOX). We unveiled unexpected amino acid cooperativity at P1-Met with P3'-His and noncanonical P1-His with P2-Phe, and the importance of the threonine trio (Thr24-Thr25-Thr26) in the prime side binding domain I in defining prime side binding in SARS-CoV-2 3CLpro. From these analyses, we rationally designed quenched-fluorescence natural amino acid peptide substrates with >15× improved sensitivity and high peptide solubility, facilitating handling and application for screening of new antiviral drugs.

Repurposing of antimycobacterium drugs for COVID-19 treatment by targeting SARS CoV-2 main protease: An in-silico perspective.

The global mortality rate has been significantly impacted by the COVID-19 pandemic, caused by the SARS CoV-2 virus. Although the pursuit for a potent antiviral is still in progress, experimental therapies based on repurposing of existing drugs is being attempted. One important therapeutic target for COVID-19 is the main protease (Mpro) that cleaves the viral polyprotein in its replication process. Recently minocycline, an antimycobacterium drug, has been successfully implemented for the treatment of COVID-19 patients. But it's mode of action is still far from clear. Furthermore, it remains unresolved whether alternative antimycobacterium drugs can effectively regulate SARS CoV-2 by inhibiting the enzymatic activity of Mpro. To comprehend these facets, eight well-established antimycobacterium drugs were put through molecular docking experiments. Four of the antimycobacterium drugs (minocycline, rifampicin, clofazimine and ofloxacin) were selected by comparing their binding affinities towards Mpro. All of the four drugs interacted with both the catalytic residues of Mpro (His41 and Cys145). Additionally, molecular dynamics experiments demonstrated that the Mpro-minocyline complex has enhanced stability, experiences reduced conformational fluctuations and greater compactness than other three Mpro-antimycobacterium and Mpro-N3/lopinavir complexes. This research furnishes evidences for implementation of minocycline against SARS CoV-2. In addition, our findings also indicate other three antimycobacterium/antituberculosis drugs (rifampicin, clofazimine and ofloxacin) could potentially be evaluated for COVID-19 therapy.

Development of a Fluorescent Assay and Imidazole-Containing Inhibitors by Targeting SARS-CoV-2 Nsp13 Helicase.

Nsp13, a non-structural protein belonging to the coronavirus family 1B (SF1B) helicase, exhibits 5'-3' polarity-dependent DNA or RNA unwinding using NTPs. Crucially, it serves as a key component of the viral replication-transcription complex (RTC), playing an indispensable role in the coronavirus life cycle and thereby making it a promising target for broad-spectrum antiviral therapies. The imidazole scaffold, known for its antiviral potential, has been proposed as a potential scaffold. In this study, a fluorescence-based assay was designed by labeling dsDNA substrates with a commercial fluorophore and monitoring signal changes upon Nsp13 helicase activity. Optimization and high-throughput screening validated the feasibility of this approach. In accordance with the structural characteristics of ADP, we employed a structural-based design strategy to synthesize three classes of imidazole-based compounds through substitution reaction. Through in vitro activity research, pharmacokinetic parameter analysis, and molecular docking simulation, we identified compounds A16 (IC50 = 1.25 µM) and B3 (IC50 = 0.98 µM) as potential lead antiviral compounds for further targeted drug research.

Structure-Based Virtual Screening for Methyltransferase Inhibitors of SARS-CoV-2 nsp14 and nsp16.

The ongoing COVID-19 pandemic still threatens human health around the world. The methyltransferases (MTases) of SARS-CoV-2, specifically nsp14 and nsp16, play crucial roles in the methylation of the N7 and 2'-O positions of viral RNA, making them promising targets for the development of antiviral drugs. In this work, we performed structure-based virtual screening for nsp14 and nsp16 using the screening workflow (HTVS, SP, XP) of Schrödinger 2019 software, and we carried out biochemical assays and molecular dynamics simulation for the identification of potential MTase inhibitors. For nsp14, we screened 239,000 molecules, leading to the identification of three hits A1-A3 showing N7-MTase inhibition rates greater than 60% under a concentration of 50 µM. For the SAM binding and nsp10-16 interface sites of nsp16, the screening of 210,000 and 237,000 molecules, respectively, from ZINC15 led to the discovery of three hit compounds B1-B3 exhibiting more than 45% of 2'-O-MTase inhibition under 50 µM. These six compounds with moderate MTase inhibitory activities could be used as novel candidates for the further development of anti-SARS-CoV-2 drugs.