Identification of Darunavir Derivatives for Inhibition of SARS-CoV-2 3CLpro.

The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CLpro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CLpro potency with EC50 values of 6.3 µM and 3.5 µM and were shown to bind to SARS-CoV-2 3CLpro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CLpro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CLpro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.

AI-Driven De Novo Design and Molecular Modeling for Discovery of Small-Molecule Compounds as Potential Drug Candidates Targeting SARS-CoV-2 Main Protease.

Over the past three years, significant progress has been made in the development of novel promising drug candidates against COVID-19. However, SARS-CoV-2 mutations resulting in the emergence of new viral strains that can be resistant to the drugs used currently in the clinic necessitate the development of novel potent and broad therapeutic agents targeting different vulnerable spots of the viral proteins. In this study, two deep learning generative models were developed and used in combination with molecular modeling tools for de novo design of small molecule compounds that can inhibit the catalytic activity of SARS-CoV-2 main protease (Mpro), an enzyme critically important for mediating viral replication and transcription. As a result, the seven best scoring compounds that exhibited low values of binding free energy comparable with those calculated for two potent inhibitors of Mpro, via the same computational protocol, were selected as the most probable inhibitors of the enzyme catalytic site. In light of the data obtained, the identified compounds are assumed to present promising scaffolds for the development of new potent and broad-spectrum drugs inhibiting SARS-CoV-2 Mpro, an attractive therapeutic target for anti-COVID-19 agents.

A new generation Mpro inhibitor with potent activity against SARS-CoV-2 Omicron variants.

Emerging SARS-CoV-2 variants, particularly the Omicron variant and its sublineages, continually threaten the global public health. Small molecule antivirals are an effective treatment strategy to fight against the virus. However, the first-generation antivirals either show limited clinical efficacy and/or have some defects in pharmacokinetic (PK) properties. Moreover, with increased use of these drugs across the globe, they face great pressure of drug resistance. We herein present the discovery and characterization of a new generation antiviral drug candidate (SY110), which is a potent and selective inhibitor of SARS-CoV-2 main protease (Mpro). This compound displayed potent in vitro antiviral activity against not only the predominant SARS-CoV-2 Omicron sublineage BA.5, but also other highly pathogenic human coronaviruses including SARS-CoV-1 and MERS-CoV. In the Omicron-infected K18-hACE2 mouse model, oral treatment with SY110 significantly lowered the viral burdens in lung and alleviated the virus-induced pathology. Importantly, SY110 possesses favorable PK properties with high oral drug exposure and oral bioavailability, and also an outstanding safety profile. Furthermore, SY110 exhibited sensitivity to several drug-resistance Mpro mutations. Collectively, this investigation provides a promising new drug candidate against Omicron and other variants of SARS-CoV-2.

Identification of SARS-CoV-2 Mpro inhibitors containing P1' 4-fluorobenzothiazole moiety highly active against SARS-CoV-2.

COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro's active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.

Identification of and Mechanistic Insights into SARS-CoV-2 Main Protease Non-Covalent Inhibitors: An In-Silico Study.

The indispensable role of the SARS-CoV-2 main protease (Mpro) in the viral replication cycle and its dissimilarity to human proteases make Mpro a promising drug target. In order to identify the non-covalent Mpro inhibitors, we performed a comprehensive study using a combined computational strategy. We first screened the ZINC purchasable compound database using the pharmacophore model generated from the reference crystal structure of Mpro complexed with the inhibitor ML188. The hit compounds were then filtered by molecular docking and predicted parameters of drug-likeness and pharmacokinetics. The final molecular dynamics (MD) simulations identified three effective candidate inhibitors (ECIs) capable of maintaining binding within the substrate-binding cavity of Mpro. We further performed comparative analyses of the reference and effective complexes in terms of dynamics, thermodynamics, binding free energy (BFE), and interaction energies and modes. The results reveal that, when compared to the inter-molecular electrostatic forces/interactions, the inter-molecular van der Waals (vdW) forces/interactions are far more important in maintaining the association and determining the high affinity. Given the un-favorable effects of the inter-molecular electrostatic interactions-association destabilization by the competitive hydrogen bond (HB) interactions and the reduced binding affinity arising from the un-compensable increase in the electrostatic desolvation penalty-we suggest that enhancing the inter-molecular vdW interactions while avoiding introducing the deeply buried HBs may be a promising strategy in future inhibitor optimization.

Acriflavine and proflavine hemisulfate as potential antivirals by targeting Mpro.

The evolving SARS-CoV-2 epidemic buffets the world, and the concerted efforts are needed to explore effective drugs. M^pro is an intriguing antiviral target for interfering with viral RNA replication and transcription. In order to get potential anti-SARS-CoV-2 agents, we established an enzymatic assay using a fluorogenic substrate to screen the inhibitors of M^pro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against M^pro with IC₅₀ of 5.60 ± 0.29 µM and 2.07 ± 0.01 µM, respectively. Further evaluation of MST assay and enzymatic kinetics experiment in vitro showed that they had a certain affinity to SARS-CoV-2 M^pro and were both non-competitive inhibitors. In addition, they inhibited about 90 % HCoV-OC43 replication in BHK-21 cells at 1 µM. Both compounds showed nano-molar activities against SARS-CoV-2 virus, which were superior to GC376 for anti-HCoV-43, and equivalent to the standard molecule remdesivir. Our study demonstrated that ACF and PRF were inhibitors of M^pro, and ACF has been previously reported as a PL^pro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of coronaviruses.

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination.

BACKGROUND: Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug design. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst the many disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potential in vitro activity of L-arginine and vitamin C against SARS-CoV-2 Mpro. METHODS: The Mpro inhibition assay was developed by cloning, expression, purification, and characterization of Mpro. Selected compounds were then screened for protease inhibition. RESULTS: L-arginine was found to be active against SARS-CoV-2 Mpro, while a vitamin C/L-arginine combination had a synergistic antiviral action against Mpro. These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C were potential Mpro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVID patients. CONCLUSIONS: The findings of the current study are important because they help to identify COVID-19 treatments that are efficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategy for COVID-19 that could be used in conjunction with pharmacological agents.

Genetically Encoded Detection of Biosynthetic Protease Inhibitors.

Proteases are an important class of drug targets that continue to drive inhibitor discovery. These enzymes are prone to resistance mutations, yet their promise for treating viral diseases and other disorders continues to grow. This study develops a general approach for detecting microbially synthesized protease inhibitors and uses it to screen terpenoid pathways for inhibitory compounds. The detection scheme relies on a bacterial two-hybrid (B2H) system that links protease inactivation to the transcription of a swappable reporter gene. This system, which can accomodate multiple biochemical outputs (i.e., luminescence and antibiotic resistance), permitted the facile incorporation of four disease-relevant proteases. A B2H designed to detect the inactivation of the main protease of severe acute respiratory syndrome coronavirus 2 enabled the identification of a terpenoid inhibitor of modest potency. An analysis of multiple pathways that make this terpenoid, however, suggested that its production was necessary but not sufficient to confer a survival advantage in growth-coupled assays. This finding highlights an important challenge associated with the use of genetic selection to search for inhibitors─notably, the influence of pathway toxicity─and underlines the value of including multiple pathways with overlapping product profiles in pathway screens. This study provides a detailed experimental framework for using microbes to screen libraries of biosynthetic pathways for targeted protease inhibitors.

Structure-Based Virtual Screening and Functional Validation of Potential Hit Molecules Targeting the SARS-CoV-2 Main Protease.

The recent global health emergency caused by the coronavirus disease 2019 (COVID-19) pandemic has taken a heavy toll, both in terms of lives and economies. Vaccines against the disease have been developed, but the efficiency of vaccination campaigns worldwide has been variable due to challenges regarding production, logistics, distribution and vaccine hesitancy. Furthermore, vaccines are less effective against new variants of the SARS-CoV-2 virus and vaccination-induced immunity fades over time. These challenges and the vaccines' ineffectiveness for the infected population necessitate improved treatment options, including the inhibition of the SARS-CoV-2 main protease (Mpro). Drug repurposing to achieve inhibition could provide an immediate solution for disease management. Here, we used structure-based virtual screening (SBVS) to identify natural products (from NP-lib) and FDA-approved drugs (from e-Drug3D-lib and Drugs-lib) which bind to the Mpro active site with high-affinity and therefore could be designated as potential inhibitors. We prioritized nine candidate inhibitors (e-Drug3D-lib: Ciclesonide, Losartan and Telmisartan; Drugs-lib: Flezelastine, Hesperidin and Niceverine; NP-lib: three natural products) and predicted their half maximum inhibitory concentration using DeepPurpose, a deep learning tool for drug-target interactions. Finally, we experimentally validated Losartan and two of the natural products as in vitro Mpro inhibitors, using a bioluminescence resonance energy transfer (BRET)-based Mpro sensor. Our study suggests that existing drugs and natural products could be explored for the treatment of COVID-19.

Reaction of 7α-bromo-6-nitrocholest-5-enes with hydrazine: Formation of steroidal pyrazolines and molecular docking against SARS-CoV-2 omicron protease.

The present work reports simple and effective protocol for preparing 6α-nitro-5α-cholestano[7α,5-cd] pyrazolines (4-7) by the reaction of 7α-bromo-6-nitrocholest-5-enes (1-3) with hydrazine hydrate under reflux [the substrate (2) gave products (5) and (6) and the later on acetylation with AC2O/Py gave (7)]. In the case of reaction of 3ß-hydroxy analogue (3) with hydrazine, however, 6α-nitro-5α-cholestano [3α,5-cd] pyrazoline (8) and 6α-nitro-3ß, 5-oxido-5ß-cholestane (9) were obtained. The probable mechanism of the formation of pyrazolines has also been outlined. In the current pandemic coronavirus disease 2019 scenario, the in-silico study was performed with reactants (1-3), their products (4-9) against SARS-CoV-2 omicron protease (PDB ID:7T9L) for knowing significant interactions between them. Docking results give information that both reactants and products have binding energies ranges from -5.7 to 7.7 kcal/mol and strong interactions with various hydrophilic and hydrophobic amino acids such as ASP, PRO, PHE, SER and LEU which are significant residues playing important role in SARS-CoV-2 Omicron main protease (Mpro).

Predicting Antiviral Resistance Mutations in SARS-CoV-2 Main Protease with Computational and Experimental Screening.

The main protease (Mpro) of SARS-CoV-2 is essential for viral replication and has been the focus of many drug discovery efforts since the start of the COVID-19 pandemic. Nirmatrelvir (NTV) is an inhibitor of SARS-CoV-2 Mpro that is used in the combination drug Paxlovid for the treatment of mild to moderate COVID-19. However, with increased use of NTV across the globe, there is a possibility that future SARS-CoV-2 lineages will evolve resistance to NTV. Early prediction and monitoring of resistance mutations could allow for measures to slow the spread of resistance and for the development of new compounds with activity against resistant strains. In this work, we have used in silico mutational scanning and inhibitor docking of Mpro to identify potential resistance mutations. Subsequent in vitro experiments revealed five mutations (N142L, E166M, Q189E, Q189I, and Q192T) that reduce the potency of NTV and of a previously identified non-covalent cyclic peptide inhibitor of Mpro. The E166M mutation reduced the half-maximal inhibitory concentration (IC50) of NTV 24-fold and 118-fold for the non-covalent peptide inhibitor. Our findings inform the ongoing genomic surveillance of emerging SARS-CoV-2 lineages.

Structural insights into Nirmatrelvir (PF-07321332)-3C-like SARS-CoV-2 protease complexation: a ligand Gaussian accelerated molecular dynamics study.

Coronavirus 3C-like protease (3CLpro) is found in SARS-CoV-2 virus, which causes COVID-19. 3CLpro controls virus replication and is a major target for target-based antiviral discovery. As reported by Pfizer, Nirmatrelvir (PF-07321332) is a competitive protein inhibitor and a clinical candidate for orally delivered medication. However, the binding mechanisms between Nirmatrelvir and 3CLpro complex structures remain unknown. This study incorporated ligand Gaussian accelerated molecular dynamics, the one-dimensional and two-dimensional potential of mean force, normal molecular dynamics, and Kramers' rate theory to determine the binding and dissociation rate constants (koff and kon) associated with the binding of the 3CLpro protein to the Nirmatrelvir inhibitor. The proposed approach addresses the challenges in designing small-molecule antiviral drugs.

Natural inhibitors of SARS-CoV-2 main protease: structure based pharmacophore modeling, molecular docking and molecular dynamic simulation studies.

Main protease (Mpro) plays a key role in replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study was designed for finding natural inhibitors of SARS-CoV-2 Mpro by in silico methods. To this end, the co-crystal structure of Mpro with telaprevir was explored and receptor-ligand pharmacophore models were developed and validated using pharmit. The database of "ZINC Natural Products" was screened, and 288 compounds were filtered according to pharmacophore features. In the next step, Lipinski's rule of five was applied and absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the filtered compounds were calculated using in silico methods. The resulted 15 compounds were docked into the active site of Mpro and those with the highest binding scores and better interaction including ZINC61991204, ZINC67910260, ZINC61991203, and ZINC08790293 were selected. Further analysis by molecular dynamic simulation studies showed that ZINC61991203 and ZINC08790293 dissociated from Mpro active site, while ZINC426421106 and ZINC5481346 were stable. Root mean square deviation (RMSD), radius of gyration (Rg), number of hydrogen bonds between ligand and protein during the time of simulation, and root mean square fluctuations (RMSF) of protein and ligands were calculated, and components of binding free energy were calculated using the molecular mechanic/Poisson-Boltzmann surface area (MM/PBSA) method. The result of all the analysis indicated that ZINC61991204 and ZINC67910260 are drug-like and nontoxic and have a high potential for inhibiting Mpro.

Adaptive Mutation in the Main Protease Cleavage Site of Feline Coronavirus Renders the Virus More Resistant to Main Protease Inhibitors.

The rapid global emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused serious health problems, highlighting the urgent need for antiviral drugs. The viral main protease (Mpro) plays an important role in viral replication and thus remains the target of choice for the prevention or treatment of several viral diseases due to high sequence and structural conservation. Prolonged use of viral protease inhibitors can lead to the development of mutants resistant to those inhibitors and to many of the available antiviral drugs. Here, we used feline infectious peritonitis virus (FIPV) as a model to investigate its development of resistance under pressure from the Mpro inhibitor GC376. Passage of wild-type (WT) FIPV in the presence of GC376 selected for a mutation in the nsp12 region where Mpro cleaves the substrate between nsp12 and nsp13. This mutation confers up to 3-fold resistance to GC376 and nirmatrelvir, as determined by EC50 assay. In vitro biochemical and cellular experiments confirmed that FIPV adapts to the stress of GC376 by mutating the nsp12 and nsp13 hydrolysis site to facilitate cleavage by Mpro and release to mediate replication and transcription. Finally, we demonstrate that GC376 cannot treat FIP-resistant mutants that cause FIP in animals. Taken together, these results suggest that Mpro affects the replication of coronaviruses (CoVs) and the drug resistance to GC376 by regulating the amount of RdRp from a distant site. These findings provide further support for the use of an antiviral drug combination as a broad-spectrum therapy to protect against contemporary and emerging CoVs. IMPORTANCE CoVs cause serious human infections, and antiviral drugs are currently approved to treat these infections. The development of protease-targeting therapeutics for CoV infection is hindered by resistance mutations. Therefore, we should pay attention to its resistance to antiviral drugs. Here, we identified possible mutations that lead to relapse after clinical treatment of FIP. One amino acid substitution in the nsp12 polymerase at the Mpro cleavage site provided low-level resistance to GC376 after selection exposure to the GC376 parental nucleoside. Resistance mutations enhanced FIPV viral fitness in vitro and attenuated the therapeutic effect of GC376 in an animal model of FIPV infection. Our research explains the evolutionary characteristics of coronaviruses under antiviral drugs, which is helpful for a more comprehensive understanding of the molecular basis of virus resistance and provides important basic data for the effective prevention and control of CoVs.

Celastrol: A lead compound that inhibits SARS-CoV-2 replication, the activity of viral and human cysteine proteases, and virus-induced IL-6 secretion.

The global emergence of coronavirus disease 2019 (COVID-19) has caused substantial human casualties. Clinical manifestations of this disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and hyperinflammation. In face of the urgent demand for effective drugs to treat COVID-19, we have searched for candidate compounds using in silico approach followed by experimental validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F, as one of the best compounds out of 39 drug candidates. Celastrol reverted the gene expression signature from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells and irreversibly inhibited the recombinant forms of the viral and human cysteine proteases involved in virus invasion, such as Mpro (main protease), PLpro (papain-like protease), and recombinant human cathepsin L. Celastrol suppressed SARS-CoV-2 replication in human and monkey cell lines and decreased interleukin-6 (IL-6) secretion in the SARS-CoV-2-infected human cell line. Celastrol acted in a concentration-dependent manner, with undetectable signs of cytotoxicity, and inhibited in vitro replication of the parental and SARS-CoV-2 variant. Therefore, celastrol is a promising lead compound to develop new drug candidates to face COVID-19 due to its ability to suppress SARS-CoV-2 replication and IL-6 production in infected cells.

Discovery of 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel inhibitors of SARS-CoV-2 main protease.

The COVID-19 posed a serious threat to human life and health, and SARS-CoV-2 Mpro has been considered as an attractive drug target for the treatment of COVID-19. Herein, we report 2-(furan-2-ylmethylene)hydrazine-1-carbothioamide derivatives as novel inhibitors of SARS-CoV-2 Mpro developed by in-house library screening and biological evaluation. Similarity search led to the identification of compound F8-S43 with the enzymatic IC50 value of 10.76 µM. Further structure-based drug design and synthetic optimization uncovered compounds F8-B6 and F8-B22 as novel non-peptidomimetic inhibitors of Mpro with IC50 values of 1.57 µM and 1.55 µM, respectively. Moreover, enzymatic kinetic assay and mass spectrometry demonstrated that F8-B6 was a reversible covalent inhibitor of Mpro. Besides, F8-B6 showed low cytotoxicity with CC50 values of more than 100 µM in Vero and MDCK cells. Overall, these novel SARS-CoV-2 Mpro non-peptidomimetic inhibitors provide a useful starting point for further structural optimization.

Anisodamine potently inhibits SARS-CoV-2 infection in vitro and targets its main protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provoked a pandemic of acute respiratory disease, namely coronavirus disease 2019 (COVID-19). Currently, effective drugs for this disease are urgently warranted. Anisodamine is a traditional Chinese medicine that is predicted as a potential therapeutic drug for the treatment of COVID-19. Therefore, this study aimed to investigate its antiviral activity and crucial targets in SARS-CoV-2 infection. SARS-CoV-2 and anisodamine were co-cultured in Vero E6 cells, and the antiviral activity of anisodamine was assessed by immunofluorescence assay. The antiviral activity of anisodamine was further measured by pseudovirus entry assay in HEK293/hACE2 cells. Finally, the predictions of crucial targets of anisodamine on SARS-CoV-2 were analyzed by molecular docking studies. We discovered that anisodamine suppressed SARS-CoV-2 infection in Vero E6 cells, and reduced the SARS-CoV-2 pseudovirus entry to HEK293/hACE2 cells. Furthermore, molecular docking studies indicated that anisodamine may target SARS-CoV-2 main protease (Mpro) with the docking score of -6.63 kcal/mol and formed three H-bonds with Gly143, Cys145, and Cys44 amino acid residues at the predicted active site of Mpro. This study suggests that anisodamine is a potent antiviral agent for treating COVID-19.

Discovery of novel SARS-CoV-2 inhibitors targeting the main protease Mpro by virtual screenings and hit optimization.

Two years after its emergence, SARS-CoV-2 still represents a serious and global threat to human health. Antiviral drug development usually takes a long time and, to increase the chances of success, chemical variability of hit compounds represents a valuable source for the discovery of new antivirals. In this work, we applied a platform of variably oriented virtual screening campaigns to seek for novel chemical scaffolds for SARS-CoV-2 main protease (Mpro) inhibitors. The study on the resulting 30 best hits led to the identification of a series of structurally unrelated Mpro inhibitors. Some of them exhibited antiviral activity in the low micromolar range against SARS-CoV-2 and other human coronaviruses (HCoVs) in different cell lines. Time-of-addition experiments demonstrated an antiviral effect during the viral replication cycle at a time frame consistent with the inhibition of SARS-CoV-2 Mpro activity. As a proof-of-concept, to validate the pharmaceutical potential of the selected hits against SARS-CoV-2, we rationally optimized one of the hit compounds and obtained two potent SARS-CoV-2 inhibitors with increased activity against Mpro both in vitro and in a cellular context, as well as against SARS-CoV-2 replication in infected cells. This study significantly contributes to the expansion of the chemical variability of SARS-CoV-2 Mpro inhibitors and provides new scaffolds to be exploited for pan-coronavirus antiviral drug development.

Reconstruction of the unbinding pathways of noncovalent SARS-CoV and SARS-CoV-2 3CLpro inhibitors using unbiased molecular dynamics simulations.

The main protease (3CLpro) is one of the essential components of the SARS-CoVs viral life cycle, which makes it an interesting target for overpowering these viruses. Although many covalent and noncovalent inhibitors have been designed to inhibit this molecular target, none have gained FDA approval as a drug. Because of the high rate of COVID-19 pandemic development, in addition to laboratory research, we require in silico methods to accelerate rational drug design. The unbinding pathways of two SARS-CoV and SARS-CoV-2 3CLpro noncovalent inhibitors with the PDB IDs: 3V3M, 4MDS, 6W63, 5RF7 were explored from a comparative perspective using unbiased molecular dynamics (UMD) simulations. We uncovered common weak points for selected inhibitors that could not interact significantly with a binding pocket at specific residues by all their fragments. So water molecules entered the free binding S regions and weakened protein-inhibitor fundamental interactions gradually. N142, G143, and H163 are the essential residues, which cause key protein-ligand interactions in the binding pocket. We believe that these results will help design new potent inhibitors against SARS-CoV-2.