A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system.

Nirmatrelvir was the first protease inhibitor specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available protease inhibitors (nirmatrelvir and ensitrelvir) with cell-based, biochemical and SARS-CoV-2 replicon assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease inhibitor resistance mechanisms and show the relevance of specific mutations, thereby informing treatment decisions.

Discovery of the potent covalent inhibitor with an acrylate warhead for SARS-CoV-2 3CL protease.

COVID-19 has caused severe consequences in terms of public health and economy worldwide since its outbreak in December 2019. SARS-CoV-2 3C-like protease (3CLpro), crucial for the viral replications, is an attractive target for the development of antiviral drugs. In this study, several kinds of Michael acceptor warheads were utilized to hunt for potent covalent inhibitors against 3CLpro. Meanwhile, novel 3CLpro inhibitors with the P3-3,5-dichloro-4-(2-(dimethylamino)ethoxy)phenyl moiety were designed and synthesized which may form salt bridge with residue Glu166. Among them, two compounds 12b and 12c exhibited high inhibitory activities against SARS-CoV-2 3CLpro. Further investigations suggested that 12b with an acrylate warhead displayed potent activity against HCoV-OC43 (EC50 = 97 nM) and SARS-CoV-2 replicon (EC50 = 45 nM) and low cytotoxicity (CC50 > 10 µM) in Huh7 cells. Taken together, this study devised two series of 3CLpro inhibitors and provided the potent SARS-CoV-2 3CLpro inhibitor (12b) which may be used for treating coronavirus infections.

A Gaussia luciferase reporter assay for the evaluation of coronavirus Nsp5/3CLpro activity.

Human coronaviruses (hCoVs) infect millions of people every year. Among these, MERS, SARS-CoV-1, and SARS-CoV-2 caused significant morbidity and mortality and their emergence highlights the risk of possible future coronavirus outbreaks. Therefore, broadly-active anti-coronavirus drugs are needed. Pharmacological inhibition of the hCoV protease Nsp5 (3CLpro) is clinically beneficial as shown by the wide and effective use of Paxlovid (nirmatrelvir, ritonavir). However, further treatment options are required due to the risk of drug resistance. To facilitate the assessment of coronavirus protease function and its pharmacological inhibition, we developed an assay allowing rapid and reliable quantification of Nsp5 activity under biosafety level 1 conditions. It is based on an ACE2-Gal4 transcription factor fusion protein separated by a Nsp5 recognition site. Cleavage by Nsp5 releases the Gal4 transcription factor, which then induces the expression of Gaussia luciferase. Our assay is compatible with Nsp5 proteases from all hCoVs and allows simultaneous measurement of inhibitory and cytotoxic effects of the tested compounds. Proof-of-concept measurements confirmed that nirmatrelvir, GC376 and lopinavir inhibit SARS-CoV-2 Nsp5 function. Furthermore, the assay accurately predicted the impact of Nsp5 mutations on catalytic activity and inhibitor sensitivity. Overall, the reporter assay is suitable for evaluating viral protease activity.

Anti- COVID-19 drug discovery by flavonoid derivatives: an extensive computational drug design approach.

The present study deals with the in-silico analyses of several flavonoid derivatives to explore COVID-19 through pharmacophore modelling, molecular docking, molecular dynamics, drug-likeness, and ADME properties. The initial literature study revealed that many flavonoids, including luteolin, quercetin, kaempferol, and baicalin may be useful against SARS ß-coronaviruses, prompting the selection of their potential derivatives to investigate their abilities as inhibitors of COVID-19. The findings were streamlined using in silico molecular docking, which revealed promising energy-binding interactions between all flavonoid derivatives and the targeted protein. Notably, compounds 8, 9, 13, and 15 demonstrated higher potency against the coronavirus Mpro protein (PDB ID 6M2N). Compound 8 has a -7.2 Kcal/mol affinity for the protein and binds to it by hydrogen bonding with Gln192 and π-sulfur bonding with Met-165. Compound 9 exhibited a significant interaction with the main protease, demonstrating an affinity of -7.9 kcal/mol. Gln-192, Glu-189, Pro-168, and His-41 were the principle amino acid residues involved in this interaction. The docking score for compound 13 is -7.5 Kcal/mol, and it binds to the protease enzyme by making interactions with Leu-41, π-sigma, and Gln-189. These interactions include hydrogen bonding and π-sulfur. The major protease and compound 15 were found to bind with a favourable affinity of -6.8 Kcal/mol. This finding was further validated through molecular dynamic simulation for 1ns, analysing parameters such as RMSD, RMSF, and RoG profiles. The RoG values for all four of the compounds varied significantly (35.2-36.4). The results demonstrated the stability of the selected compounds during the simulation. After passing the stability testing, the compounds underwent screening for ADME and drug-likeness properties, fulfilling all the necessary criteria. The findings of the study may support further efforts for the discovery and development of safe drugs to treat COVID-19.

Structural and virologic mechanism of the emergence of resistance to Mpro inhibitors in SARS-CoV-2.

We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (Mpro) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2WK521WT in VeroE6TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2WK521E166V), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of Mpro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1'-Glu166 interactions. TKB245 stayed bound to MproE166V, whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of Mpro, and compromised the enzymatic activity; the Ki values of recombinant MproE166V for nirmatrelvir and TKB245 were 117±3 and 17.1±1.9 µM, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to MproE166V than nirmatrelvir. SARS-CoV-2WK521WT selected with 5h acquired A191T substitution in Mpro (SARS-CoV-2WK521A191T) and better replicated in the presence of 5h, than SARS-CoV-2WK521WT. However, no significant enzymatic or structural changes in MproA191T were observed. The replicability of SARS-CoV-2WK521E166V proved to be compromised compared to SARS-CoV-2WK521WT but predominated over SARS-CoV-2WK521WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2WK521A191T surpassed that of SARS-CoV-2WK521WT in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2's drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.

Nonpeptidic Irreversible Inhibitors of SARS-CoV-2 Main Protease with Potent Antiviral Activity.

SARS-CoV-2 infections pose a high risk for vulnerable patients. In this study, we designed benzoic acid halopyridyl esters bearing a variety of substituents as irreversible inhibitors of the main viral protease (Mpro). Altogether, 55 benzoyl chloro/bromo-pyridyl esters were synthesized, with broad variation of the substitution pattern on the benzoyl moiety. A workflow was employed for multiparametric optimization, including Mpro inhibition assays of SARS-CoV-2 and related pathogenic coronaviruses, the duration of enzyme inhibition, the compounds' stability versus glutathione, cytotoxicity, and antiviral activity. Several compounds showed IC50 values in the low nanomolar range, kinact/Ki values of >100,000 M-1 s-1 and high antiviral activity. High-resolution X-ray cocrystal structures indicated an important role of ortho-fluorobenzoyl substitution, forming a water network that stabilizes the inhibitor-bound enzyme. The most potent antiviral compound was the p-ethoxy-o-fluorobenzoyl chloropyridyl ester (PSB-21110, 29b, MW 296 g/mol; EC50 2.68 nM), which may serve as a lead structure for broad-spectrum anticoronaviral therapeutics.

Assay Development and Validation for Innovative Antiviral Development Targeting the N-Terminal Autoprocessing of SARS-CoV-2 Main Protease Precursors.

The SARS-CoV-2 main protease (Mpro) is initially synthesized as part of polyprotein precursors that undergo autoproteolysis to release the free mature Mpro. To investigate the autoprocessing mechanism in transfected mammalian cells, we examined several fusion precursors, with the mature SARS-CoV-2 Mpro along with the flanking amino acids (to keep the native substrate sequences) sandwiched between different tags. Our analyses revealed differential proteolysis kinetics at the N- and C-terminal cleavage sites. Particularly, N-terminal processing is differentially influenced by various upstream fusion tags (GST, sGST, CD63, and Nsp4) and amino acid variations at the N-terminal P1 position, suggesting that precursor catalysis is flexible and subject to complex regulation. Mutating Q to E at the N-terminal P1 position altered both precursor catalysis and the properties of the released Mpro. Interestingly, the wild-type precursors exhibited different enzymatic activities compared to those of the released Mpro, displaying much lower susceptibility to known inhibitors targeting the mature form. These findings suggest the precursors as alternative targets for antiviral development. Accordingly, we developed and validated a high-throughput screening (HTS)-compatible platform for functional screening of compounds targeting either the N-terminal processing of the SARS-CoV-2 Mpro precursor autoprocessing or the released mature Mpro through different mechanisms of action.

Discovery of SARS-CoV-2 papain-like protease (PLpro) inhibitors with efficacy in a murine infection model.

Vaccines and first-generation antiviral therapeutics have provided important protection against COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there remains a need for additional therapeutic options that provide enhanced efficacy and protection against potential viral resistance. The SARS-CoV-2 papain-like protease (PLpro) is one of the two essential cysteine proteases involved in viral replication. While inhibitors of the SARS-CoV-2 main protease have demonstrated clinical efficacy, known PLpro inhibitors have, to date, lacked the inhibitory potency and requisite pharmacokinetics to demonstrate that targeting PLpro translates to in vivo efficacy in a preclinical setting. Here, we report the machine learning-driven discovery of potent, selective, and orally available SARS-CoV-2 PLpro inhibitors, with lead compound PF-07957472 (4) providing robust efficacy in a mouse-adapted model of COVID-19 infection.

An ISG15-Based High-Throughput Screening Assay for Identification and Characterization of SARS-CoV-2 Inhibitors Targeting Papain-like Protease.

Emergence of newer variants of SARS-CoV-2 underscores the need for effective antivirals to complement the vaccination program in managing COVID-19. The multi-functional papain-like protease (PLpro) of SARS-CoV-2 is an essential viral protein that not only regulates the viral replication but also modulates the host immune system, making it a promising therapeutic target. To this end, we developed an in vitro interferon stimulating gene 15 (ISG15)-based Förster resonance energy transfer (FRET) assay and screened the National Cancer Institute (NCI) Diversity Set VI compound library, which comprises 1584 small molecules. Subsequently, we assessed the PLpro enzymatic activity in the presence of screened molecules. We identified three potential PLpro inhibitors, namely, NSC338106, 651084, and 679525, with IC50 values in the range from 3.3 to 6.0 µM. These molecules demonstrated in vitro inhibition of the enzyme activity and exhibited antiviral activity against SARS-CoV-2, with EC50 values ranging from 0.4 to 4.6 µM. The molecular docking of all three small molecules to PLpro suggested their specificity towards the enzyme's active site. Overall, our study contributes promising prospects for further developing potential antivirals to combat SARS-CoV-2 infection.

SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, Mpro (also called 3C-like protease, 3CLpro), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, Mpro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified Mpro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported Mpro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known Mpro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.

4-(3-Phenylsulfonylindol-2-yl)-1-(pyridin-2-yl)piperazinyl-methanones as Potent Inhibitors of both SARS-CoV-2 and HCoV-OC43 Viruses.

SARS-CoV-2 and HCoV-OC43 belong to the same ß genus of the Coronaviridae family. SARS-CoV-2 was responsible for the recent COVID-19 pandemic, and HCoV-OC43 is the etiological agent of mild upper respiratory tract infections. SARS-COV-2 and HCoV-OC43 co-infections were found in children with respiratory symptoms during the COVID-19 pandemic. The two ß-coronaviruses share a high degree of homology between the 3CLpro active sites, so much so that the safer HCoV-OC43 has been suggested as a tool for the identification of new anti-SARS-COV-2 agents. Compounds 5 and 24 inhibited effectively both Wuhan and British SARS-CoV-2 patient isolates in Vero E6 cells and the HCoV-OC43 in MRC-5 cells at low micromolar concentrations. The inhibition was apparently exerted via targeting the 3CLpro active sites of both viruses. Compounds 5 and 24 at 100 µM inhibited the SARS-CoV-2 3CLpro activity of 61.78 and 67.30%, respectively. These findings highlight 5 and 24 as lead compounds of a novel class of antiviral agents with the potential to treat SARS-COV-2 and HCoV-OC43 infections.

Development of a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro.

The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

New combined Inverse-QSAR and molecular docking method for scaffold-based drug discovery.

Computer-aided drug discovery plays a vital role in developing novel medications for various diseases. The COVID-19 pandemic has heightened the need for innovative approaches to design lead compounds with the potential to become effective drugs. Specifically, designing promising inhibitors of the SARS-CoV-2 main protease (Mpro) is crucial, as it plays a key role in viral replication. Phytochemicals, primarily flavonoids and flavonols from medicinal plants, were screened. Fifty small molecules were selected for molecular docking analysis against SARS-CoV-2 Mpro (PDB ID: 6LU7). Binding energies and interactions were analyzed and compared to those of the anti-SARS-CoV-2 inhibitor Nirmatrelvir. Using these 50 structures as a training set, a QSAR model was built employing simple, reversible topological descriptors. An inverse-QSAR analysis was then performed on 29 = 512 hydroxyl combinations at nine possible positions on the flavone and flavonol scaffold. The model predicted three novel, promising compounds exhibiting the most favorable binding energies (-8.5 kcal/mol) among the 512 possible hydroxyl combinations: 3,6,7,2',4'-pentahydroxyflavone (PF9), 6,7,2',4'-tetrahydroxyflavone (PF11), and 3,6,7,4'-tetrahydroxyflavone (PF15). Molecular dynamics (MD) simulations demonstrated the stability of the PF9/Mpro complex over 300 ns of simulation. These predicted structures, reported here for the first time, warrant synthesis and further evaluation of their biological activity through in vitro and in vivo studies.

Disrupting protease and deubiquitinase activities of SARS-CoV-2 papain-like protease by natural and synthetic products discovered through multiple computational and biochemical approaches.

The single-stranded RNA genome of SARS-CoV-2 encodes several structural and non-structural proteins, among which the papain-like protease (PLpro) is crucial for viral replication and immune evasion and has emerged as a promising therapeutic target. The current study aims to discover new inhibitors of PLpro that can simultaneously disrupt its protease and deubiquitinase activities. Using multiple computational approaches, six compounds (CP1-CP6) were selected from our in-house compounds database, with higher docking scores (-7.97 kcal/mol to -8.14 kcal/mol) and fitted well in the active pocket of PLpro. Furthermore, utilizing microscale molecular dynamics simulations (MD), the dynamic behavior of selected compounds was studied. Those molecules strongly binds at the PLpro active site and forms stable complexes. The dynamic motions suggest that the binding of CP1-CP6 brought the protein to a closed conformational state, thereby altering its normal function. In an in vitro evaluation, CP2 showed the most significant inhibitory potential for PLpro (protease activity = 2.71 ± 0.33 µM and deubiquitinase activity = 3.11 ± 0.75 µM), followed by CP1, CP5, CP4 and CP6. Additionally, CP1-CP6 showed no cytotoxicity at a concentration of 30 µM in the human BJ cell line.

Discovery of macrocyclic covalent inhibitors for severe acute respiratory syndrome coronavirus 2 3CL protease.

The coronavirus disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spread worldwide for more than 3 years. Although the hospitalization rate and mortality have decreased dramatically due to wide vaccination effort and improved treatment options, the disease is still a global health issue due to constant viral mutations, causing negative impact on social and economic activities. In addition, long COVID and complications arising from COVID-19 weeks after infection have become a concern for public health experts. Therefore, better treatments for COVID-19 are still needed. Herein, we describe a class of macrocyclic peptidomimetic compounds that are potent inhibitors of SARS-Cov-2 3CL protease (3CLpro). Significantly, some of the compounds showed a higher stability against human liver microsomes (HLM t1/2 > 180 min) and may be suitable for oral administration without the need for a pharmacokinetic (PK) boosting agent such as ritonavir.

Dual SARS-CoV-2 and MERS-CoV inhibitors from Artemisia monosperma: isolation, structure elucidation, molecular modelling studies, and in vitro activities.

The COVID-19 pandemic has spread throughout the whole globe, so it is imperative that all available resources be used to treat this scourge. In reality, the development of new pharmaceuticals has mostly benefited from natural products. The widespread medicinal usage of species in the Asteraceae family is extensively researched. In this study, compounds isolated from methanolic extract of Artemisia monosperma Delile, a wild plant whose grows in Egypt's Sinai Peninsula. Three compounds, stigmasterol 3-O-ß-D-glucopyranoside 1, rhamnetin 3, and padmatin 6, were first isolated from this species. In addition, five previously reported compounds, arcapillin 2, jaceosidin 4, hispidulin 5, 7-O-methyleriodictyol 7, and eupatilin 8, were isolated. Applying molecular modelling simulations revealed two compounds, arcapillin 2 and rhamnetin 3 with the best docking interactions and energies within SARS-CoV-2 Mpro-binding site (-6.16, and -6.70 kcal mol-1, respectively). The top-docked compounds (2-3) were further evaluated for inhibitory concentrations (IC50), and half-maximal cytotoxicity (CC50) of both SARS-CoV-2 and MERS-CoV. Interestingly, arcapillin showed high antiviral activity towards SARS-CoV-2 and MERS-CoV, with IC50 values of 190.8 µg mL-1 and 16.58 µg mL-1, respectively. These findings may hold promise for further preclinical and clinical research, particularly on arcapillin itself or in collaboration with other drugs for COVID-19 treatment.

Covalent small-molecule inhibitors of SARS-CoV-2 Mpro: Insights into their design, classification, biological activity, and binding interactions.

Since 2020, many compounds have been investigated for their potential use in the treatment of SARS-CoV-2 infection. Among these agents, a huge number of natural products and FDA-approved drugs have been evaluated as potential therapeutics for SARS-CoV-2 using virtual screening and docking studies. However, the identification of the molecular targets involved in viral replication led to the development of rationally designed anti-SARS-CoV-2 agents. Among these targets, the main protease (Mpro) is one of the key enzymes needed in the replication of the virus. The data gleaned from the crystal structures of SARS-CoV-2 Mpro complexes with small-molecule covalent inhibitors has been used in the design and discovery of many highly potent and broad-spectrum Mpro inhibitors. The current review focuses mainly on the covalent type of SARS-CoV-2 Mpro inhibitors. The design, chemistry, and classification of these inhibitors were also in focus. The biological activity of these inhibitors, including their inhibitory activities against Mpro, their antiviral activities, and the SAR studies, were discussed. The review also describes the potential mechanism of the interaction between these inhibitors and the catalytic Cys145 residue in Mpro. Moreover, the binding modes and key binding interactions of these covalent inhibitors were also illustrated. The covalent inhibitors discussed in this review were of diverse chemical nature and origin. Their antiviral activity was mediated mainly by the inhibition of SARS-CoV-2 Mpro, with IC50 values in the micromolar to the nanomolar range. Many of these inhibitors exhibited broad-spectrum inhibitory activity against the Mpro enzymes of other coronaviruses (SARS-CoV-1 and MERS-CoV). The dual inhibition of the Mpro and PLpro enzymes of SARS-CoV-2 could also provide higher therapeutic benefits than Mpro inhibition. Despite the approval of nirmatrelvir by the FDA, many mutations in the Mpro enzyme of SARS-CoV-2 have been reported. Although some of these mutations did not affect the potency of nirmatrelvir, there is an urgent need to develop a second generation of Mpro inhibitors. We hope that the data summarized in this review could help researchers in the design of a new potent generation of SARS-CoV-2 Mpro inhibitors.

The small molecule inhibitor of SARS-CoV-2 3CLpro EDP-235 prevents viral replication and transmission in vivo.

The COVID-19 pandemic has led to the deaths of millions of people and severe global economic impacts. Small molecule therapeutics have played an important role in the fight against SARS-CoV-2, the virus responsible for COVID-19, but their efficacy has been limited in scope and availability, with many people unable to access their benefits, and better options are needed. EDP-235 is specifically designed to inhibit the SARS-CoV-2 3CLpro, with potent nanomolar activity against all SARS-CoV-2 variants to date, as well as clinically relevant human and zoonotic coronaviruses. EDP-235 maintains potency against variants bearing mutations associated with nirmatrelvir resistance. Additionally, EDP-235 demonstrates a ≥ 500-fold selectivity index against multiple host proteases. In a male Syrian hamster model of COVID-19, EDP-235 suppresses SARS-CoV-2 replication and viral-induced hamster lung pathology. In a female ferret model, EDP-235 inhibits production of SARS-CoV-2 infectious virus and RNA at multiple anatomical sites. Furthermore, SARS-CoV-2 contact transmission does not occur when naïve ferrets are co-housed with infected, EDP-235-treated ferrets. Collectively, these results demonstrate that EDP-235 is a broad-spectrum coronavirus inhibitor with efficacy in animal models of primary infection and transmission.

Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad-spectrum antiviral compounds.

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach-HyCoSuL-to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.

On the dual role of (+)-catechin as primary antioxidant and inhibitor of viral proteases.

Natural antioxidants have become the subject of many investigations due to the role that they play in the reduction of oxidative stress. Their main scavenging mechanisms concern the direct inactivation of free radicals and the coordination of metal ions involved in Fenton-like reactions. Recently, increasing attention has been paid to non-covalent inhibition of enzymes involved in different diseases by the antioxidants. Here, a computational investigation on the primary antioxidant power of (+)-catechin against the •OOH radical has been performed in both lipid-like and aqueous environments, taking into account the relevant species present in the simulated acid-base equilibria at the physiological pH. Hydrogen Atom Transfer (HAT), Single Electron Transfer (SET), and Radical Adduct Formation (RAF) mechanisms were studied, and relative rate constants were estimated. The potential inhibitory activity of the (+)-catechin towards the most important proteases from SARS-CoV-2, 3C-like (Mpro) and papain-like (PLpro) proteases was also investigated by MD simulations to provide deeper atomistic insights on the binding sites. Based on the antioxidant and antiviral properties also unravelled by comparison with other molecules having similar chemical scaffold, our results propose that (+)-CTc satisfies can explicate a dual action as antioxidant and antiviral in particular versus Mpro from SARS-CoV-2.