Disulfiram inhibits coronaviral main protease by conjugating to its substrate entry site.

Coronaviruses (CoV) are highly pathogenic single-strand RNA viruses. CoV infections cause fatal respiratory symptoms and lung injuries in humans and significant economic losses in livestock. Since the SARS-2 outbreak in 2019, the highly conserved main protease (Mpro), also termed 3-chymotrypsin-like protease (3CLpro), has been considered an attractive drug target for treating CoV infections. Mpro mediates the proteolytic cleavage of eleven sites in viral polypeptides necessary for virus replication. Here, we report that disulfiram, an FDA-approved drug for alcoholic treatment, exhibits a broad-spectrum inhibitory effect on CoV Mpros. Analytical ultracentrifugation and circular dichroism analyses indicated that disulfiram treatment blocks the dimeric formation of SARS and PEDV Mpros and decreases the thermostability of SARS, SARS-2, and PEDV Mpros, whereas it facilitates the dimerization and stability of MERS Mpro. Furthermore, mass spectrometry and structural alignment revealed that disulfiram targets the Cys44 residue of Mpros, which is located at the substrate entrance and close to the catalytic His41. In addition, molecular docking analysis suggests that disulfiram conjugation interferes with substrate entry to the catalytic center. In agreement, mutation of Cys44 modulates the disulfiram sensitivity of CoV Mpros. Our study suggests a broad-spectrum inhibitory function of disulfiram against CoV Mpros.

Highly specific SARS-CoV-2 main protease (Mpro) mutations against the clinical antiviral ensitrelvir selected in a safe, VSV-based system.

In the SARS-CoV-2 pandemic, the so far two most effective approved antivirals are the protease inhibitors nirmatrelvir, in combination with ritonavir (Paxlovid) and ensitrelvir (Xocova). However, antivirals and indeed all antimicrobial drugs are sooner or later challenged by resistance mutations. Studying such mutations is essential for treatment decisions and pandemic preparedness. At the same time, generating resistant viruses to assess mutants is controversial, especially with pathogens of pandemic potential like SARS-CoV-2. To circumvent gain-of-function research with non-attenuated SARS-CoV-2, a previously developed safe system based on a chimeric vesicular stomatitis virus dependent on the SARS-CoV-2 main protease (VSV-Mpro) was used to select mutations against ensitrelvir. Ensitrelvir is clinically especially relevant due to its single-substance formulation, avoiding drug-drug interactions by the co-formulated CYP3A4 inhibitor ritonavir in Paxlovid. By treating VSV-Mpro with ensitrelvir, several highly-specific resistant mutants against this inhibitor were selected, while being still fully or largely susceptible to nirmatrelvir. We then confirmed several ensitrelvir-specific mutants in gold standard enzymatic assays and SARS-CoV-2 replicons. These findings indicate that the two inhibitors can have distinct viral resistance profiles, which could determine treatment decisions.

SARS-CoV-2 replication and drug discovery.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions of people and continues to wreak havoc across the globe. This sudden and deadly pandemic emphasizes the necessity for anti-viral drug development that can be rapidly administered to reduce morbidity, mortality, and virus propagation. Thus, lacking efficient anti-COVID-19 treatment, and especially given the lengthy drug development process as well as the critical death tool that has been associated with SARS-CoV-2 since its outbreak, drug repurposing (or repositioning) constitutes so far, the ideal and ready-to-go best approach in mitigating viral spread, containing the infection, and reducing the COVID-19-associated death rate. Indeed, based on the molecular similarity approach of SARS-CoV-2 with previous coronaviruses (CoVs), repurposed drugs have been reported to hamper SARS-CoV-2 replication. Therefore, understanding the inhibition mechanisms of viral replication by repurposed anti-viral drugs and chemicals known to block CoV and SARS-CoV-2 multiplication is crucial, and it opens the way for particular treatment options and COVID-19 therapeutics. In this review, we highlighted molecular basics underlying drug-repurposing strategies against SARS-CoV-2. Notably, we discussed inhibition mechanisms of viral replication, involving and including inhibition of SARS-CoV-2 proteases (3C-like protease, 3CLpro or Papain-like protease, PLpro) by protease inhibitors such as Carmofur, Ebselen, and GRL017, polymerases (RNA-dependent RNA-polymerase, RdRp) by drugs like Suramin, Remdesivir, or Favipiravir, and proteins/peptides inhibiting virus-cell fusion and host cell replication pathways, such as Disulfiram, GC376, and Molnupiravir. When applicable, comparisons with SARS-CoV inhibitors approved for clinical use were made to provide further insights to understand molecular basics in inhibiting SARS-CoV-2 replication and draw conclusions for future drug discovery research.

Glycyrrhetinic acid blocks SARS-CoV-2 infection by activating the cGAS-STING signalling pathway.

BACKGROUND AND PURPOSE: Traditional Chinese medicine (TCM) played an important role in controlling the COVID-19 pandemic, but the scientific basis and its active ingredients are still weakly studied. This study aims to decipher the underlying anti-SARS-CoV-2 mechanisms of glycyrrhetinic acid (GA). EXPERIMENTAL APPROACH: GA's anti-SARS-CoV-2 effect was verified both in vitro and in vivo. Homogeneous time-resolved fluorescence assays, biolayer interferometry technology, and molecular docking were employed to examine interactions of GA with human stimulator of interferon genes (hSTING). Immunofluorescence staining, western blot, and RT-qPCR were used to investigate nuclear translocation of interferon regulatory factor 3 (IRF3) and levels of STING target genes. Pharmacokinetics of GA was studied in mice. KEY RESULTS: GA could directly bind to Ser162 and Tyr240 residues of hSTING, thus up-regulating downstream targets and activation of the STING signalling pathway. Such activation is crucial for limiting the replication of SARS-CoV-2 Omicron in Calu-3 cells and protecting against lung injury induced by SARS-CoV-2 Omicron infection in K18-ACE2 transgenic mice. Immunofluorescence staining and western blot indicated that GA increased levels of phosphorylated STING, phosphorylated TANK-binding kinase-1, and cyclic GMP-AMP synthase (cGAS). Importantly, GA increased nuclear translocation of IRF3. Pharmacokinetic analysis of GA in mice indicated it can be absorbed into circulation and detected in the lung at a stable level. CONCLUSION AND IMPLICATIONS: Activation of the cGAS-STING pathway through the GA-STING-IRF3 axis is essential for the antiviral activity of GA in mice, providing new insights into the potential translation of GA for treating SARS-CoV-2 in patients.

Synthesis and biological evaluation of novel peptidomimetic inhibitors of the coronavirus 3C-like protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and related variants, are responsible for the devastating coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 main protease (Mpro) plays a central role in the replication of the virus and represents an attractive drug target. Herein, we report the discovery of novel SARS-CoV-2 Mpro covalent inhibitors, including highly effective compound NIP-22c which displays high potency against several key variants and clinically relevant nirmatrelvir Mpro E166V mutants.

Discovery of natural catechol derivatives as covalent SARS-CoV-2 3CLpro inhibitors.

The COVID-19 pandemic caused by SARS-CoV-2 continues to pose a threat to public health, and extensive research by scientists worldwide has also prompted the development of antiviral therapies. The 3C-like protease (3CLpro) is critical for SARS-CoV-2 replication and acts as an effective target for drug development. To date, numerous of natural products have been reported to exhibit inhibitory effects on 3CLpro, which encourages us to identify other novel inhibitors and elucidate their mechanism of action. In this study, we first screened an in-house compound library of 101 natural products using FRET assay, and found that oleuropein showed good inhibitory activity against SARS CoV-2 3CLpro with an IC50 value of 4.18 µM. Further studies revealed that the catechol core is essential for activity and can covalently bind to SARS-CoV-2 3CLpro. Among other 45 catechol derivatives, wedelolactone, capsazepine and brazilin showed better SARS-CoV-2 3CLpro inhibitory activities with IC50 values of 1.35 µM, 1.95 µM and 1.18 µM, respectively. These catechol derivatives were verified to be irreversible covalent inhibitors by time-dependent experiments, enzymatic kinetic studies, dilution and dialysis assays. It also exhibited good selectivity towards different cysteine proteases (SARS-CoV-2 PLpro, cathepsin B and cathepsin L). Subsequently, the binding affinity between brazilin and SARS-CoV-2 3CLpro was determined by SPR assay with KD value of 0.80 µM. Molecular dynamic (MD) simulations study showed the binding mode of brazilin in the target protein. In particular, brazilin displayed good anti-SARS-CoV-2 activity in A549-hACE2-TMPRSS2 cells with EC50 values of 7.85 ± 0.20 µM and 5.24 ± 0.21 µM for full time and post-infection treatments, respectively. This study provides a promising lead compound for the development of novel anti-SARS-CoV-2 drugs.

Methyl rosmarinate is an allosteric inhibitor of SARS-CoV-2 3 CL protease as a potential candidate against SARS-cov-2 infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been ongoing for more than three years and urgently needs to be addressed. Traditional Chinese medicine (TCM) prescriptions have played an important role in the clinical treatment of patients with COVID-19 in China. However, it is difficult to uncover the potential molecular mechanisms of the active ingredients in these TCM prescriptions. In this paper, we developed a new approach by integrating the experimental assay, virtual screening, and the experimental verification, exploring the rapid discovery of active ingredients from TCM prescriptions. To achieve this goal, 4 TCM prescriptions in clinical use for different indications were selected to find the antiviral active ingredients in TCMs. The 3-chymotrypsin-like protease (3CLpro), an important target for fighting COVID-19, was utilized to determine the inhibitory activity of the TCM prescriptions and single herb. It was found that 10 single herbs had better inhibitory activity than other herbs by using a fluorescence resonance energy transfer (FRET) - based enzymatic assay of SARS-CoV-2 3CLpro. The ingredients contained in 10 herbs were thus virtually screened and the predicted active ingredients were experimentally validated. Thus, such a research strategy firstly removed many single herbs with no inhibitory activity against SARS-CoV-2 3CLpro at the very beginning by FRET-based assay, making our subsequent virtual screening more effective. Finally, 4 active components were found to have stronger inhibitory effects on SARS-CoV-2 3CLpro, and their inhibitory mechanism was subsequently investigated. Among of them, methyl rosmarinate as an allosteric inhibitor of SARS-CoV-2 3CLpro was confirmed and its ability to inhibit viral replication was demonstrated by the SARS-CoV-2 replicon system. To validate the binding mode via docking, the mutation experiment, circular dichroism (CD), enzymatic inhibition and surface plasmon resonance (SPR) assay were performed, demonstrating that methyl rosmarinate bound to the allosteric site of SARS-CoV-2 3CLpro. In conclusion, this paper provides the new ideas for the rapid discovery of active ingredients in TCM prescriptions based on a specific target, and methyl rosmarinate has the potential to be developed as an antiviral therapeutic candidate against SARS-CoV-2 infection.

Exploration of the P1 residue in 3CL protease inhibitors leading to the discovery of a 2-tetrahydrofuran P1 replacement.

The virally encoded 3C-like protease (3CLpro) is a well-validated drug target for the inhibition of coronaviruses including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Most inhibitors of 3CLpro are peptidomimetic, with a γ-lactam in place of Gln at the P1 position of the pseudopeptide chain. An effort was pursued to identify a viable alternative to the γ-lactam P1 mimetic which would improve physicochemical properties while retaining affinity for the target. Discovery of a 2-tetrahydrofuran as a suitable P1 replacement that is a potent enzymatic inhibitor of 3CLpro in SARS-CoV-2 virus is described herein.

Design, synthesis and biological evaluation of biaryl amide derivatives against SARS-CoV-2 with dual-target mechanism.

The COVID-19 pandemic highlights the urgent need to develop effective small-molecule antivirals. Thirty-three novel biaryl amide derivatives were synthesized and evaluated for anti-coronaviral activity. Some significant SARs were uncovered and the intensive structure modifications led to the most active compounds 8b and 8h. The broad-spectrum anti-coronaviral effects of 8h were validated at RNA and protein levels. 8h inhibits coronavirus replication at multiple stages, from virus entry to virus dsRNA synthesis. The mechanism of action showed that 8h may simultaneously act on 3CLpro and TMPRSS2 to display anti-coronaviral effects. 8h combined with RdRp inhibitor showed synergistic inhibitory activity against coronavirus. This study confirmed that biaryl amide derivatives may be a new class of potential therapeutic agents against coronavirus with multiple target effect, worthy of further investigation.

Danshensu inhibits SARS-CoV-2 by targeting its main protease as a specific covalent inhibitor and discovery of bifunctional compounds eliciting antiviral and anti-inflammatory activity.

The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a serious threat to human. Since there are still no effective treatment options against the new emerging variants of SARS-CoV-2, it is necessary to devote a continuous endeavor for more targeted drugs and the preparation for the next pandemic. Salvia miltiorrhiza and its active ingredients possess wide antiviral activities, including against SARS-CoV-2. Danshensu, as one of the most important active ingredients in Salvia miltiorrhiza, has been reported to inhibit the entry of SARS-CoV-2 into ACE2 (angiotensin-converting enzyme 2)-overexpressed HEK-293T cells and Vero-E6 cells. However, there is a paucity of information regarding its detailed target and mechanism against SARS-CoV-2. Here, we present Danshensu as a covalent inhibitor of 3-chymotrypsin-like protease (3CLpro) against SARS-CoV-2 by the time-dependent inhibition assay (TDI) and mass spectrometry analysis. Further molecular docking, site-directed mutagenesis, circular dichroism (CD) and fluorescence spectra revealed that Danshensu covalently binds to C145 of SARS-CoV-2 3CLpro, meanwhile forming the hydrogen bonds with S144, H163 and E166 in the S1 site. Structure-based optimization of Danshensu led to the discovery of the promising compounds with good inhibitory activity and microsomal stability in vitro. Due to Danshensu inhibiting lung inflammation in the mouse model, we found that Danshensu derivatives also showed better anti-inflammatory activity than Danshensu in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Thus, our study provides not only the clue of the efficacy of Salvia miltiorrhiza against SARS-CoV-2, but also a detailed mechanistic insight into the covalent mode of action of Danshensu for design of covalent inhibitors against SARS-CoV-2 3CLpro, highlighting its potential as a bifunctional molecule with antivirus and anti-inflammation.

Chrysin 7-O-ß-D-glucuronide, a dual inhibitor of SARS-CoV-2 3CLpro and PLpro, for the prevention and treatment of COVID-19.

The emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) resulted in the coronavirus disease 2019 (COVID-19) pandemic. Given the advent of subvariants, there is an urgent need to develop novel drugs. The aim of this study was to find SARS-CoV-2 inhibitors from Scutellaria baicalensis Georgi targeting the proteases 3CLpro and PLpro. After screening 25 flavonoids, chrysin 7-O-ß-D-glucuronide was found to be a potent inhibitor of SARS-CoV-2 on Vero E6 cells, with half-maximal effective concentration of 8.72 µM. Surface plasmon resonance assay, site-directed mutagenesis and enzymatic activity measurements indicated that chrysin-7-O-ß-D-glucuronide inhibits SARS-CoV-2 by binding to H41 of 3CLpro, and K157 and E167 of PLpro. Hydrogen-deuterium exchange mass spectrometry analysis showed that chrysin-7-O-ß-D-glucuronide changes the conformation of PLpro. Finally, chrysin 7-O-ß-D-glucuronide was shown to have anti-inflammatory activity, mainly due to reduction of the levels of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6.

Development of de-novo coronavirus 3-chymotrypsin-like protease (3CLpro) inhibitors since COVID-19 outbreak: A strategy to tackle challenges of persistent virus infection.

Although no longer a public health emergency of international concern, COVID-19 remains a persistent and critical health concern. The development of effective antiviral drugs could serve as the ultimate piece of the puzzle to curbing this global crisis. 3-chymotrypsin-like protease (3CLpro), with its substrate specificity mirroring that of the main picornavirus 3C protease and conserved across various coronaviruses, emerges as an ideal candidate for broad-spectrum antiviral drug development. Moreover, it holds the potential as a reliable contingency option to combat emerging SARS-CoV-2 variants. In this light, the approved drugs, promising candidates, and de-novo small molecule therapeutics targeting 3CLpro since the COVID-19 outbreak in 2020 are discussed. Emphasizing the significance of diverse structural characteristics in inhibitors, be they peptidomimetic or nonpeptidic, with a shared mission to minimize the risk of cross-resistance. Moreover, the authors propose an innovative optimization strategy for 3CLpro reversible covalent PROTACs, optimizing pharmacodynamics and pharmacokinetics to better prepare for potential future viral outbreaks.

In Silico Screening of Natural Flavonoids against 3-Chymotrypsin-like Protease of SARS-CoV-2 Using Machine Learning and Molecular Modeling.

The "Long-COVID syndrome" has posed significant challenges due to a lack of validated therapeutic options. We developed a novel multi-step virtual screening strategy to reliably identify inhibitors against 3-chymotrypsin-like protease of SARS-CoV-2 from abundant flavonoids, which represents a promising source of antiviral and immune-boosting nutrients. We identified 57 interacting residues as contributors to the protein-ligand binding pocket. Their energy interaction profiles constituted the input features for Machine Learning (ML) models. The consensus of 25 classifiers trained using various ML algorithms attained 93.9% accuracy and a 6.4% false-positive-rate. The consensus of 10 regression models for binding energy prediction also achieved a low root-mean-square error of 1.18 kcal/mol. We screened out 120 flavonoid hits first and retained 50 drug-like hits after predefined ADMET filtering to ensure bioavailability and safety profiles. Furthermore, molecular dynamics simulations prioritized nine bioactive flavonoids as promising anti-SARS-CoV-2 agents exhibiting both high structural stability (root-mean-square deviation < 5 Å for 218 ns) and low MM/PBSA binding free energy (<-6 kcal/mol). Among them, KB-2 (PubChem-CID, 14630497) and 9-O-Methylglyceofuran (PubChem-CID, 44257401) displayed excellent binding affinity and desirable pharmacokinetic capabilities. These compounds have great potential to serve as oral nutraceuticals with therapeutic and prophylactic properties as care strategies for patients with long-COVID syndrome.

Discovery of highly potent covalent SARS-CoV-2 3CLpro inhibitors bearing 2-sulfoxyl-1,3,4-oxadiazole scaffold for combating COVID-19.

The coronavirus disease (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has emerged as a major public health crisis, posing a significant threat to human well-being. Despite the availability of vaccines, COVID-19 continues to spread owing to the emergence of SARS-CoV-2 mutants. This highlights the urgent need for the discovery of more effective drugs to combat COVID-19. As an important target for COVID-19 treatment, 3C-like protease (3CLpro) plays a crucial role in the replication of SARS-CoV-2. In our previous research, we demonstrated the potent inhibitory activities of compound A1, which contains a 2-sulfonyl-1,3,4-oxadiazole scaffold, against SARS-CoV-2 3CLpro. Herein, we present a detailed investigation of structural optimization of A1 and conduct a study on the structure-activity relationship. Among the various compounds tested, sulfoxide D6 demonstrates a potent irreversible inhibitory activity (IC50 = 0.030 µM) against SARS-CoV-2 3CLpro, as well as a favorable selectivity towards host cysteine proteases such as cathepsin B and cathepsin L. Utilizing mass spectrometry-based peptide profiling, we found that D6 covalently binds to Cys145 of SARS-CoV-2 3CLpro. Some representative compounds, namely C11, D9 and D10 also demonstrates antiviral activity against SARS-CoV-2 in Vero E6 cells. Overall, the investigation of the 2-sulfoxyl-1,3,4-oxadiazole scaffold as a novel cysteine reactive warhead would provide valuable insights into the design of potent covalent 3CLpro inhibitors for COVID-19 treatment.

Elucidation of novel compounds and epitope-based peptide vaccine design against C30 endopeptidase regions of SARS-CoV-2 using immunoinformatics approaches.

There has been progressive improvement in immunoinformatics approaches for epitope-based peptide design. Computational-based immune-informatics approaches were applied to identify the epitopes of SARS-CoV-2 to develop vaccines. The accessibility of the SARS-CoV-2 protein surface was analyzed, and hexa-peptide sequences (KTPKYK) were observed having a maximum score of 8.254, located between amino acids 97 and 102, whereas the FSVLAC at amino acids 112 to 117 showed the lowest score of 0.114. The surface flexibility of the target protein ranged from 0.864 to 1.099 having amino acid ranges of 159 to 165 and 118 to 124, respectively, harboring the FCYMHHM and YNGSPSG hepta-peptide sequences. The surface flexibility was predicted, and a 0.864 score was observed from amino acids 159 to 165 with the hepta-peptide (FCYMHHM) sequence. Moreover, the highest score of 1.099 was observed between amino acids 118 and 124 against YNGSPSG. B-cell epitopes and cytotoxic T-lymphocyte (CTL) epitopes were also identified against SARS-CoV-2. In molecular docking analyses, -0.54 to -26.21 kcal/mol global energy was observed against the selected CTL epitopes, exhibiting binding solid energies of -3.33 to -26.36 kcal/mol. Based on optimization, eight epitopes (SEDMLNPNY, GSVGFNIDY, LLEDEFTPF, DYDCVSFCY, GTDLEGNFY, QTFSVLACY, TVNVLAWLY, and TANPKTPKY) showed reliable findings. The study calculated the associated HLA alleles with MHC-I and MHC-II and found that MHC-I epitopes had higher population coverage (0.9019% and 0.5639%) than MHC-II epitopes, which ranged from 58.49% to 34.71% in Italy and China, respectively. The CTL epitopes were docked with antigenic sites and analyzed with MHC-I HLA protein. In addition, virtual screening was conducted using the ZINC database library, which contained 3,447 compounds. The 10 top-ranked scrutinized molecules (ZINC222731806, ZINC077293241, ZINC014880001, ZINC003830427, ZINC030731133, ZINC003932831, ZINC003816514, ZINC004245650, ZINC000057255, and ZINC011592639) exhibited the least binding energy (-8.8 to -7.5 kcal/mol). The molecular dynamics (MD) and immune simulation data suggest that these epitopes could be used to design an effective SARS-CoV-2 vaccine in the form of a peptide-based vaccine. Our identified CTL epitopes have the potential to inhibit SARS-CoV-2 replication.

Design, synthesis and biological evaluation of peptidomimetic benzothiazolyl ketones as 3CLpro inhibitors against SARS-CoV-2.

A series of peptidomimetic compounds containing benzothiazolyl ketone and [2.2.1] azabicyclic ring was designed, synthesized and evaluated in the hope of obtaining potent oral 3CLpro inhibitors with improved pharmacokinetic properties. Among the target compounds, 11b had the best enzymatic potency (IC50 = 0.110 µM) and 11e had the best microsomal stability (t1/2 > 120 min) and good enzyme activity (IC50 = 0.868 µM). Therefore, compounds 11b and 11e were chosen for further evaluation of pharmacokinetics in ICR mice. The results exhibited that the AUC(0-t) of 11e was 5143 h*ng/mL following single-dose oral administration of 20 mg/kg, and the F was 67.98%. Further structural modification was made to obtain compounds 11g-11j based on 11e. Among them, 11j exhibited the best enzyme inhibition activity against SARS-CoV-2 3CLpro (IC50 = 1.646 µM), the AUC(0-t) was 32473 h*ng/mL (20 mg/kg, po), and the F was 48.1%. In addition, 11j displayed significant anti-SARS-CoV-2 activity (EC50 = 0.18 µM) and low cytotoxicity (CC50 > 50 µM) in Vero E6 cells. All of the above results suggested that compound 11j was a promising lead compound in the development of oral 3CLpro inhibitors and deserved further research.

Structure-guided design of direct-acting antivirals that exploit the gem-dimethyl effect and potently inhibit 3CL proteases of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and middle east respiratory syndrome coronavirus (MERS-CoV).

The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CLpro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 µM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1eand1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies.

High throughput screening for drugs that inhibit 3C-like protease in SARS-CoV-2.

The SARS coronavirus 2 (SARS-CoV-2) pandemic remains a major problem in many parts of the world and infection rates remain at extremely high levels. This high prevalence drives the continued emergence of new variants, and possibly ones that are more vaccine-resistant and that can drive infections even in highly vaccinated populations. The high rate of variant evolution makes clear the need for new therapeutics that can be clinically applied to minimize or eliminate the effects of COVID-19. With a hurdle of 10 years, on average, for first in class small molecule therapeutics to achieve FDA approval, the fastest way to identify therapeutics is by drug repurposing. To this end, we developed a high throughput cell-based screen that incorporates the essential viral 3C-like protease and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or FDA-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Medicinal chemistry efforts to optimize the hits identified Tranilast as a potential lead. Here, we report the rapid screening and identification of potentially relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral 3C-like protease.

Discovery, synthesis and mechanism study of 2,3,5-substituted [1,2,4]-thiadiazoles as covalent inhibitors targeting 3C-Like protease of SARS-CoV-2.

The 3C-like protease (3CLpro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CLpro as non-peptidomimetic covalent binders at submicromolar levels, with IC50 values ranging from 0.118 to 0.582 µM. Interestingly, these compounds were also shown to inhibit PLpro with the same level of IC50 values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC50 value of 7.249 µM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a, one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study.

Ensitrelvir is effective against SARS-CoV-2 3CL protease mutants circulating globally.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a public health concern worldwide. Ensitrelvir (S-217622) has been evaluated as an antiviral treatment for COVID-19, targeting SARS-CoV-2 3C-like protease (3CLpro). Ensitrelvir has been reported to have comparable antiviral activity against some of the SARS-CoV-2 variants: alpha, beta, gamma, delta, and omicron (BA.1.18). In this paper, we describe that ensitrelvir is effective against newly emerging SARS-CoV-2 variants and globally prevalent 3CLpro mutations. Ensitrelvir exhibited comparable antiviral activity against SARS-CoV-2 variants, including recently emerging ones: omicron (BA1.1, BA.2, BA.2.75, BA.4, BA.5, BQ.1.1, XBB.1, and XE), mu, lambda, and theta. Genetic surveillance of SARS-CoV-2 3CLpro, the target of ensitrelvir, was conducted using a public database and identified 11 major 3CLpro mutations circulating globally (G15S, T21I, T24I, K88R, L89F, K90R, P108S, P132H, A193V, H246Y, and A255V). The 3CLpro mutation from proline to histidine at amino acid position 132 was especially identified in the omicron variant, with prevalence of 99.69%. Enzyme kinetic assay revealed that these 3CLpro mutants have enzymatic activity comparable to that of the wild type (WT). Next, we assessed the inhibitory effect of ensitrelvir against mutated 3CLpro, with it showing inhibitory effects similar to that against the WT. These in vitro data suggest that ensitrelvir will be effective against currently circulating SARS-CoV-2 variants, including omicron variants and those carrying 3CLpro mutations, which emerging novel SARS-CoV-2 variants could carry.