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.

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.

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.

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.

Improved SARS-CoV-2 main protease high-throughput screening assay using a 5-carboxyfluorescein substrate.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a global threat to human health has highlighted the need for the development of novel therapies targeting current and emerging coronaviruses with pandemic potential. The coronavirus main protease (Mpro, also called 3CLpro) is a validated drug target against coronaviruses and has been heavily studied since the emergence of SARS-CoV-2 in late 2019. Here, we report the biophysical and enzymatic characterization of native Mpro, then characterize the steady-state kinetics of several commonly used FRET substrates, fluorogenic substrates, and six of the 11 reported SARS-CoV-2 polyprotein cleavage sequences. We then assessed the suitability of these substrates for high-throughput screening. Guided by our assessment of these substrates, we developed an improved 5-carboxyfluorescein-based FRET substrate, which is better suited for high-throughput screening and is less susceptible to interference and false positives than existing substrates. This study provides a useful framework for the design of coronavirus Mpro enzyme assays to facilitate the discovery and development of therapies targeting Mpro.

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.

Discovery and mechanism of action of Thonzonium bromide from an FDA-approved drug library with potent and broad-spectrum inhibitory activity against main proteases of human coronaviruses.

Although the effective drugs or vaccines have been developed to prevent the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), their efficacy may be limited for the viral evolution and immune escape. Thus, it is urgently needed to develop the novel broad-spectrum antiviral agents to control the coronavirus disease 2019 (COVID-19) global pandemic. The 3C-like protease (3CLpro) is a highly conserved cysteine proteinase that plays a pivotal role in processing the viral polyprotein to create non-structural proteins (nsps) for replication and transcription of SARS-CoV-2, making it an attractive antiviral target for developing broad-spectrum antiviral agents against SARS-CoV-2. In this study, we identified Thonzonium bromide as an inhibitor of SARS-CoV-2 3CLpro with an IC50 value of 2.04 ± 0.25 µM by fluorescence resonance energy transfer (FRET)-based enzymatic inhibition assay from the FDA-approved drug library. Next, we determined the inhibitory activity of Thonzonium bromide analogues against SARS-CoV-2 3CLpro and analyzed their structure-activity relationship (SAR). Interestingly, Thonzonium bromide showed better inhibitory activity than other analogues. Further fluorescence quenching assay, enzyme kinetics analysis, circular dichroism (CD) analysis and molecular docking studies showed that Thonzonium bromide inhibited SARS-CoV-2 3CLpro activity by firmly occupying the catalytic site and inducing conformational changes of the protease. In addition, Thonzonium bromide didn't exhibit inhibitory activity on human chymotrypsin C (CTRC) and Dipeptidyl peptidase IV (DPP-IV), indicating that it had a certain selectivity. Finally, we measured the inhibitory activities of Thonzonium bromide against 3CLpro of SARS-CoV, MERS-CoV and HCoV-229E and found that it had the broad-spectrum inhibitory activity against the proteases of human coronaviruses. These results provide the possible mechanism of action of Thonzonium bromide, highlighting its potential efficacy against multiple human coronaviruses.

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.

Identification of Aloe-derived natural products as prospective lead scaffolds for SARS-CoV-2 main protease (Mpro) inhibitors.

In the past two years, the COVID-19 pandemic has caused over 5 million deaths and 250 million infections worldwide. Despite successful vaccination efforts and emergency approval of small molecule therapies, a diverse range of antivirals is still needed to combat the inevitable resistance that will arise from new SARS-CoV-2 variants. The main protease of SARS-CoV-2 (Mpro) is an attractive drug target due to the clinical success of protease inhibitors against other viruses, such as HIV and HCV. However, in order to combat resistance, various chemical scaffolds need to be identified that have the potential to be developed into potent inhibitors. To this end, we screened a high-content protease inhibitor library against Mproin vitro, in order to identify structurally diverse compounds that could be further developed into antiviral leads. Our high-content screening efforts retrieved 27 hits each with > 50% inhibition in our Mpro FRET assay. Of these, four of the top inhibitor compounds were chosen for follow-up due to their potency and drugability (Lipinski's rules of five criteria): anacardic acid, aloesin, aloeresin D, and TCID. Further analysis via dose response curves revealed IC50 values of 6.8 µM, 38.9 µM, 125.3 µM, and 138.0 µM for each compound, respectively. Molecular docking studies demonstrated that the four inhibitors bound at the catalytic active site of Mpro with varying binding energies (-7.5 to -5.6 kcal/mol). Furthermore, Mpro FRET assay kinetic studies demonstrated that Mpro catalysis is better represented by a sigmoidal Hill model than the standard Michaelis-Menten hyperbola, indicating substantial cooperativity of the active enzyme dimer. This result suggests that the dimerization interface could be an attractive target for allosteric inhibitors. In conclusion, we identified two closely-related natural product compounds from the Aloe plant (aloesin and aloeresin D) that may serve as novel scaffolds for Mpro inhibitor design and additionally confirmed the strongly cooperative kinetics of Mpro proteolysis. These results further advance our knowledge of structure-function relationships in Mpro and offer new molecular scaffolds for inhibitor design.

Novel inhibitors of the main protease enzyme of SARS-CoV-2 identified via molecular dynamics simulation-guided in vitro assay.

For the COVID-19 pandemic caused by SARS-CoV-2, there are currently no effective drugs or vaccines to treat this coronavirus infection. In this study, we focus on the main protease enzyme of SARS-CoV-2, 3CLpro, which is critical for viral replication. We employ explicit solvent molecular dynamics simulations of about 150 compounds docked into 3CLpro's binding site and that had emerged as good main protease ligands from our previous in silico screening of over 1.2 million compounds. By incoporating protein dynamics and applying a range of structural descriptors, such as the ability to form specific contacts with the catalytic dyad residues of 3CLpro and the structural fluctuations of the ligands in the binding site, we are able to further refine our compound selection. Fourteen compounds including estradiol shown to be the most promising based on our calculations were procured and screened against recombinant 3CLpro in a fluorescence assay. Eight of these compounds have significant activity in inhibiting the SARS-CoV-2 main protease. Among these are corilagin, a gallotannin, and lurasidone, an antipsychotic drug, which emerged as the most promising natural product and drug, respectively, and might thus be candidates for drug repurposing for the treatment of COVID-19. In addition, we also tested the inhibitory activity of testosterone, and our results reveal testosterone as possessing moderate inhibitory potency against the 3CLpro enzyme, which may thus provide an explanation why older men are more severely affected by COVID-19.

A network pharmacology based approach for predicting active ingredients and potential mechanism of Lianhuaqingwen capsule in treating COVID-19.

The outbreak of severe respiratory disease caused by SARS-CoV-2 has led to millions of infections and raised global health concerns. Lianhuaqingwen capsule (LHQW-C), a traditional Chinese medicine (TCM) formula widely used for respiratory diseases, shows therapeutic efficacy in the application of coronavirus disease 2019 (COVID-19). However, the active ingredients, drug targets, and the therapeutic mechanisms of LHQW-C in treating COVID-19 are poorly understood. In this study, an integrating network pharmacology approach including pharmacokinetic screening, target prediction (targets of the host and targets from the SARS-CoV-2), network analysis, GO enrichment analysis, KEGG pathway enrichment analysis, and virtual docking were conducted. Finally, 158 active ingredients in LHQW-C were screen out, and 49 targets were predicted. GO function analysis revealed that these targets were associated with inflammatory response, oxidative stress reaction, and other biological processes. KEGG enrichment analysis indicated that the targets of LHQW-C were highly enriched to several immune response-related and inflammation-related pathways, including the IL-17 signaling pathway, TNF signaling pathway, NF-kappa B signaling pathway, and Th17 cell differentiation. Moreover, four key components (quercetin, luteolin, wogonin, and kaempferol) showed a high binding affinity with SARS-CoV-2 3-chymotrypsin-like protease (3CL pro). The study indicates that some anti-inflammatory ingredients in LHQW-C probably modulate the inflammatory response in severely ill patients with COVID-19.

A Review of the Current Landscape of SARS-CoV-2 Main Protease Inhibitors: Have We Hit the Bullseye Yet?

In this review, we collected 1765 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M-pro inhibitors from the bibliography and other sources, such as the COVID Moonshot project and the ChEMBL database. This set of inhibitors includes only those compounds whose inhibitory capacity, mainly expressed as the half-maximal inhibitory concentration (IC50) value, against M-pro from SARS-CoV-2 has been determined. Several covalent warheads are used to treat covalent and non-covalent inhibitors separately. Chemical space, the variation of the IC50 inhibitory activity when measured by different methods or laboratories, and the influence of 1,4-dithiothreitol (DTT) are discussed. When available, we have collected the values of inhibition of viral replication measured with a cellular antiviral assay and expressed as half maximal effective concentration (EC50) values, and their possible relationship to inhibitory potency against M-pro is analyzed. Finally, the most potent covalent and non-covalent inhibitors that simultaneously inhibit the SARS-CoV-2 M-pro and the virus replication in vitro are discussed.

Combining Different Docking Engines and Consensus Strategies to Design and Validate Optimized Virtual Screening Protocols for the SARS-CoV-2 3CL Protease.

The 3CL-Protease appears to be a very promising medicinal target to develop anti-SARS-CoV-2 agents. The availability of resolved structures allows structure-based computational approaches to be carried out even though the lack of known inhibitors prevents a proper validation of the performed simulations. The innovative idea of the study is to exploit known inhibitors of SARS-CoV 3CL-Pro as a training set to perform and validate multiple virtual screening campaigns. Docking simulations using four different programs (Fred, Glide, LiGen, and PLANTS) were performed investigating the role of both multiple binding modes (by binding space) and multiple isomers/states (by developing the corresponding isomeric space). The computed docking scores were used to develop consensus models, which allow an in-depth comparison of the resulting performances. On average, the reached performances revealed the different sensitivity to isomeric differences and multiple binding modes between the four docking engines. In detail, Glide and LiGen are the tools that best benefit from isomeric and binding space, respectively, while Fred is the most insensitive program. The obtained results emphasize the fruitful role of combining various docking tools to optimize the predictive performances. Taken together, the performed simulations allowed the rational development of highly performing virtual screening workflows, which could be further optimized by considering different 3CL-Pro structures and, more importantly, by including true SARS-CoV-2 3CL-Pro inhibitors (as learning set) when available.

Review on the potential action mechanisms of Chinese medicines in treating Coronavirus Disease 2019 (COVID-19).

The Coronavirus Disease 2019 (COVID-19) has been declared as a global pandemic, but specific medicines and vaccines are still being developed. In China, interventional therapies with traditional Chinese medicine for COVID-19 have achieved significant clinical efficacies, but the underlying pharmacological mechanisms are still unclear. This article reviewed the etiology of COVID-19 and clinical efficacy. Both network pharmacological study and literature search were used to demonstrate the possible action mechanisms of Chinese medicines in treating COVID-19. We found that Chinese medicines played the role of antivirus, anti-inflammation and immunoregulation, and target organs protection in the management of COVID-19 by multiple components acting on multiple targets at multiple pathways. AEC2 and 3CL protein could be the direct targets for inhibiting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Quercetin, kaempferol, luteolin, isorhamnetin, baicalein, naringenin, and wogonin could be the main active ingredients of Chinese medicines for the management of COVID-19 by targeting on AEC2 and 3CL protein and inhibiting inflammatory mediators, regulating immunity, and eliminating free radicals through COX-2, CASP3, IL-6, MAPK1, MAPK14, MAPK8, and REAL in the signaling pathways of IL-17, arachidonic acid, HIF-1, NF-κB, Ras, and TNF. This study may provide meaningful and useful information on further research to investigate the action mechanisms of Chinese medicines against SARS-CoV-2 and also provide a basis for sharing the "China scheme" for COVID-19 treatment.

Identification of potential binders of the main protease 3CLpro of the COVID-19 via structure-based ligand design and molecular modeling.

We have applied a computational strategy, using a combination of virtual screening, docking and molecular dynamics techniques, aimed at identifying possible lead compounds for the non-covalent inhibition of the main protease 3CLpro of the SARS-CoV2 Coronavirus. Based on the X-ray structure (PDB code: 6LU7), ligands were generated using a multimodal structure-based design and then docked to the monomer in the active state. Docking calculations show that ligand-binding is strikingly similar in SARS-CoV and SARS-CoV2 main proteases. The most potent docked ligands are found to share a common binding pattern with aromatic moieties connected by rotatable bonds in a pseudo-linear arrangement.

Prediction of Novel Inhibitors of the Main Protease (M-pro) of SARS-CoV-2 through Consensus Docking and Drug Reposition.

Since the outbreak of the COVID-19 pandemic in December 2019 and its rapid spread worldwide, the scientific community has been under pressure to react and make progress in the development of an effective treatment against the virus responsible for the disease. Here, we implement an original virtual screening (VS) protocol for repositioning approved drugs in order to predict which of them could inhibit the main protease of the virus (M-pro), a key target for antiviral drugs given its essential role in the virus' replication. Two different libraries of approved drugs were docked against the structure of M-pro using Glide, FRED and AutoDock Vina, and only the equivalent high affinity binding modes predicted simultaneously by the three docking programs were considered to correspond to bioactive poses. In this way, we took advantage of the three sampling algorithms to generate hypothetic binding modes without relying on a single scoring function to rank the results. Seven possible SARS-CoV-2 M-pro inhibitors were predicted using this approach: Perampanel, Carprofen, Celecoxib, Alprazolam, Trovafloxacin, Sarafloxacin and ethyl biscoumacetate. Carprofen and Celecoxib have been selected by the COVID Moonshot initiative for in vitro testing; they show 3.97 and 11.90% M-pro inhibition at 50 µM, respectively.

Identification, synthesis and evaluation of SARS-CoV and MERS-CoV 3C-like protease inhibitors.

Severe acute respiratory syndrome (SARS) led to a life-threatening form of atypical pneumonia in late 2002. Following that, Middle East Respiratory Syndrome (MERS-CoV) has recently emerged, killing about 36% of patients infected globally, mainly in Saudi Arabia and South Korea. Based on a scaffold we reported for inhibiting neuraminidase (NA), we synthesized the analogues and identified compounds with low micromolar inhibitory activity against 3CL(pro) of SARS-CoV and MERS-CoV. Docking studies show that a carboxylate present at either R(1) or R(4) destabilizes the oxyanion hole in the 3CL(pro). Interestingly, 3f, 3g and 3m could inhibit both NA and 3CL(pro) and serve as a starting point to develop broad-spectrum antiviral agents.

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings.

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K(i) value of 11.1 µM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.

N-acylbenzimidazoles as selective Acylators of the catalytic cystein of the coronavirus 3CL protease.

The 3CL protease (3CLpro, Mpro) plays a key role in the replication of the SARS-CoV-2 and was validated as therapeutic target by the development and approval of specific antiviral drugs (nirmatrelvir, ensitrelvir), inhibitors of this protease. Moreover, its high conservation within the coronavirus family renders it an attractive therapeutic target for the development of anti-coronavirus compounds with broad spectrum activity to control COVID-19 and future coronavirus diseases. Here we report on the design, synthesis and structure-activity relationships of a new series of small covalent reversible inhibitors of the SARS-CoV-2 3CLpro. As elucidated thanks to the X-Ray structure of some inhibitors with the 3CLpro, the mode of inhibition involves acylation of the thiol of the catalytic cysteine. The synthesis of 60 analogs led to the identification of compound 56 that inhibits the SARS-CoV-2 3CLpro with high potency (IC50 = 70 nM) and displays antiviral activity in cells (EC50 = 3.1 µM). Notably, compound 56 inhibits the 3CLpro of three other human coronaviruses and exhibit a good selectivity against two human cysteine proteases. These results demonstrate the potential of this electrophilic N-acylbenzimidazole series as a basis for further optimization.

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.