Search

New lead drugs to treat COVID-19 are beginning to emerge.

The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In this study, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 μM and 9.0 μM. In addition, three compounds inhibited PLpro with IC50 ranging from 1.9 μM to 3.3 μM. To verify the specificity of Mpro and PLpro inhibitors, our experiments included an assessment of common causes of false positives such as aggregation, high compound fluorescence, and inhibition by enzyme oxidation. Altogether, we confirmed novel classes of specific Mpro and PLpro inhibitors. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.

Existing tools to detect and visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suffer from low selectivity, poor cell permeability, and high cytotoxicity. Here we report a novel self-immolative fluorescent probe (MP590) for the highly selective and sensitive detection of the SARS-CoV-2 main protease (Mpro). This fluorescent probe was prepared by connecting a Mpro-cleavable peptide (N-acetyl-Abu-Tle-Leu-Gln) with a fluorophore (i.e., resorufin) via a self-immolative aromatic linker. Fluorescent titration results show that MP590 can detect Mpro with a limit of detection (LoD) of 35 nM and is selective over interferents such as hemoglobin, bovine serum albumin (BSA), thrombin, amylase, SARS-CoV-2 papain-like protease (PLpro), and trypsin. The cell imaging data indicate that this probe can report Mpro in HEK 293T cells transfected with a Mpro expression plasmid as well as in TMPRSS2-VeroE6 cells infected with SARS-CoV-2. Our results suggest that MP590 can both measure and monitor Mpro activity and quantitatively evaluate Mpro inhibition in infected cells, making it an important tool for diagnostic and therapeutic research on SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a serious threat to human health and lacks an effective treatment. There is an urgent need for both real-time tracking and precise treatment of the SARS-CoV-2-infected cells to mitigate and ultimately prevent viral transmission. However, selective triggering and tracking of the therapeutic process in the infected cells remains challenging. Here, we report a main protease (Mpro)-responsive, mitochondrial-targeting, and modular-peptide-conjugated probe (PSGMR) for selective imaging and inhibition of SARS-CoV-2-infected cells via enzyme-instructed self-assembly and aggregation-induced emission (AIE) effect. The amphiphilic PSGMR was constructed with tunable structure and responsive efficiency and validated with recombinant proteins, cells transfected with Mpro plasmid or infected by SARS-CoV-2, and a Mpro inhibitor. By rational construction of AIE luminogen (AIEgen) with modular peptides and Mpro, we verified that the cleavage of PSGMR yielded gradual aggregation with bright fluorescence and enhanced cytotoxicity to induce mitochondrial interference of the infected cells. This strategy may have value for selective detection and treatment of SARS-CoV-2-infected cells.

The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (Mpro). FRET-based screening against recombinant SARS-CoV-2 Mpro identified six compounds that inhibit proteolysis with nanomolar IC50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 Mpro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit Mpro competitively. Lead E24 inhibited viral replication with a nanomolar EC50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of Mpro inhibition that should facilitate the design of future COVID-19 treatments.

SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug development. In this study, we present an antiviral screening strategy involving a novel in-cell protease assay, antiviral and biochemical activity assessments, as well as structural determinations for rapid identification of protease inhibitors with low cytotoxicity. We identified eight compounds with anti-SARS-CoV-2 activity from a library of 64 repurposed drugs and modeled at protease active sites by in silico docking. We demonstrate that Sitagliptin and Daclatasvir inhibit PLpro, and MG-101, Lycorine HCl, and Nelfinavir mesylate inhibit Mpro of SARS-CoV-2. The X-ray crystal structure of Mpro in complex with MG-101 shows a covalent bond formation between the inhibitor and the active site Cys145 residue indicating its mechanism of inhibition is by blocking the substrate binding at the active site. Thus, we provide methods for rapid and effective screening and development of inhibitors for blocking virus polyprotein processing as SARS-CoV-2 antivirals. Additionally, we show that the combined inhibition of Mpro and PLpro is more effective in inhibiting SARS-CoV-2 and the delta variant.

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), threatens global public health. The world needs rapid development of new antivirals and vaccines to control the current pandemic and to control the spread of the variants. Among the proteins synthesized by the SARS-CoV-2 genome, main protease (Mpro also known as 3CLpro) is a primary drug target, due to its essential role in maturation of the viral polyproteins. In this study, we provide crystallographic evidence, along with some binding assay data, that three clinically approved anti hepatitis C virus drugs and two other drug-like compounds covalently bind to the Mpro Cys145 catalytic residue in the active site. Also, molecular docking studies can provide additional insight for the design of new antiviral inhibitors for SARS-CoV-2 using these drugs as lead compounds. One might consider derivatives of these lead compounds with higher affinity to the Mpro as potential COVID-19 therapeutics for further testing and possibly clinical trials.

This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (Mpro). The cyclic peptide inhibitor is designed to mimic the conformation of a substrate at a C-terminal autolytic cleavage site of Mpro. The cyclic peptide contains a [4-(2-aminoethyl)phenyl]-acetic acid (AEPA) linker that is designed to enforce a conformation that mimics a peptide substrate of Mpro. In vitro evaluation of the cyclic peptide inhibitor reveals that the inhibitor exhibits modest activity against Mpro and does not appear to be cleaved by the enzyme. Conformational searching predicts that the cyclic peptide inhibitor is fairly rigid, adopting a favorable conformation for binding to the active site of Mpro. Computational docking to the SARS-CoV-2 Mpro suggests that the cyclic peptide inhibitor can bind the active site of Mpro in the predicted manner. Molecular dynamics simulations provide further insights into how the cyclic peptide inhibitor may bind the active site of Mpro. Although the activity of the cyclic peptide inhibitor is modest, its design and study lays the groundwork for the development of additional cyclic peptide inhibitors against Mpro with improved activities.

COVID-19, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has presented a serious risk to global public health. The viral main protease Mpro (also called 3Clpro) encoded by NSP5 is an enzyme essential for viral replication. However, very few host proteins have been experimentally validated as targets of 3Clpro. Here, through bioinformatics analysis of 300 interferon stimulatory genes (ISGs) based on the prediction method NetCorona, we identify RNF20 (Ring Finger Protein 20) as a novel target of 3Clpro. We have also provided evidence that 3Clpro, but not the mutant 3ClproC145A without catalytic activity, cleaves RNF20 at a conserved Gln521 across species, which subsequently prevents SREBP1 from RNF20-mediated degradation and promotes SARS-CoV-2 replication. We show that RNA interference (RNAi)-mediated depletion of either RNF20 or RNF40 significantly enhances viral replication, indicating the antiviral role of RNF20/RNF40 complex against SARS-CoV-2. The involvement of SREBP1 in SARS-CoV-2 infection is evidenced by a decrease of viral replication in the cells with SREBP1 knockdown and inhibitor AM580. Taken together, our findings reveal RNF20 as a novel host target for SARS-CoV-2 main protease and indicate that 3Clpro inhibitors may treat COVID-19 through not only blocking viral polyprotein cleavage but also enhancing host antiviral response.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration-approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [Ki] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (Ki = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (Ki = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.

This paper describes the structure-based design of a preliminary drug candidate against COVID-19 using free software and publicly available X-ray crystallographic structures. The goal of this tutorial is to disseminate skills in structure-based drug design and to allow others to unleash their own creativity to design new drugs to fight the current pandemic. The tutorial begins with the X-ray crystallographic structure of the main protease (Mpro) of the SARS coronavirus (SARS-CoV) bound to a peptide substrate and then uses the UCSF Chimera software to modify the substrate to create a cyclic peptide inhibitor within the Mpro active site. Finally, the tutorial uses the molecular docking software AutoDock Vina to show the interaction of the cyclic peptide inhibitor with both SARS-CoV Mpro and the highly homologous SARS-CoV-2 Mpro. The supporting information provides an illustrated step-by-step protocol, as well as a video showing the inhibitor design process, to help readers design their own drug candidates for COVID-19 and the coronaviruses that will cause future pandemics. An accompanying preprint in bioRxiv [https://doi.org/10.1101/2020.08.03.234872] describes the synthesis of the cyclic peptide and the experimental validation as an inhibitor of SARS-CoV-2 Mpro.

Since its outbreak, the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has impacted the quality of life and cost hundreds-of-thousands of lives worldwide. Based on its global spread and mortality, there is an urgent need for novel treatments which can combat this disease. To date, the 3-chymotrypsin-like protease (3CLpro ), which is also known as the main protease, is considered among the most important pharmacological targets. The vast majority of investigated 3CLpro inhibitors are organic, non-covalent binders. Herein, the use of inorganic, coordinate covalent binders is proposed that can attenuate the activity of the protease. ReI tricarbonyl complexes were identified that demonstrate coordinate covalent enzymatic inhibition of 3CLpro . Preliminary studies indicate the selective inhibition of 3CLpro over several human proteases. This study presents the first example of metal complexes as inhibitors for the 3CLpro cysteine protease.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549/ACE2 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.

The respiratory disease COVID-19 is caused by the coronavirus SARS-CoV-2. Here we report the discovery of ethacridine as a potent drug against SARS-CoV-2 (EC50 ~ 0.08 μM). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescence assay. Plaque assays, RT-PCR and immunofluorescence imaging at various stages of viral infection demonstrate that the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Consistently, ethacridine is effective in various cell types, including primary human nasal epithelial cells that are cultured in an air-liquid interface. Taken together, our work identifies a promising, potent, and new use of the old drug via a distinct mode of action for inhibiting SARS-CoV-2.

Background: In early 2020, COVID-19 pandemic has mobilized researchers in finding new remedies including repurposing of medicinal plant products focusing on direct-acting antiviral and host-directed therapies. In this study, we performed an in vitro investigation on the standardized Marantodes pumilum extract (SKF7 ® ) focusing on anti-SARS-CoV-2 and anti-inflammatory activities., Methods: Anti-SARS-CoV-2 potential of the SKF7 ® was evaluated in SARS-CoV-2-infected Vero E6 cells and SARS-CoV-2-infected A549 cells by cytopathic effect-based assay and RT-qPCR, respectively. Target based assays were performed on the SKF7 ® against the S1-ACE2 interaction and 3CL protease activities. Anti-inflammatory activity of the SKF7 ® was evaluated by nitric oxide inhibitory and TLR2/TLR4 receptor blocker assays., Results: The SKF7 ® inhibited wild-type Wuhan (EC 50 of 21.99 µg/mL) and omicron (EC 50 of 16.29 µg/mL) SARS-CoV-2 infections in Vero-E6 cells. The SKF7 ® also inhibited the wild-type SARS-CoV-2 infection in A549 cells (EC 50 value of 6.31 µg/mL). The SKF7 ® prominently inhibited 3CL protease activity. The SKF7 ® inhibited the LPS induced-TLR4 response with the EC 50 of 16.19 µg/mL., Conclusions: In conclusion, our in vitro study highlighted anti-SARS-CoV-2 and anti-inflammatory potentials of the SKF7 ® . Future pre-clinical in vivo studies focusing on antiviral and immunomodulatory potentials of the SKF7 ® in affecting the COVID-19 pathogenesis are warranted., (© 2024. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the latest arisen contagious respiratory pathogen related to the global outbreak of atypical pneumonia pandemic (COVID-19). The essential oils (EOs) of Eucalyptus camaldulensis, E. ficifolia F. Muell., E. citriodora Hook, E. globulus Labill, E. sideroxylon Cunn. ex Woolls, and E. torquata Luehm. were investigated for its antiviral activity against SARS-CoV-2. The EOs phytochemical composition was determined using GC/MS analysis. Correlation with the explored antiviral activity was also studied using multi-variate data analysis and Pearson's correlation. The antiviral MTT and cytopathic effect inhibition assays revealed very potent and promising anti SARS-CoV-2 potential for E. citriodora EO (IC 50  = 0.00019 µg/mL and SI = 26.27). The multivariate analysis revealed α-pinene, α-terpinyl acetate, globulol, γ -terpinene, and pinocarvone were the main biomarkers for E. citriodora oil. Pearson's correlation revealed that globulol is the top positively correlated compound in E. citriodora oil to its newly explored potent anti SARS-CoV-2 potential. A molecular simulation was performed on globulol via docking in the main active sites of both SARS-CoV-2 viral main protease (Mpro) and spike protein (S). In silico predictive ADMET study was also developed to investigate the pharmacokinetic profile and predict globulol toxicity. The obtained in silico, in vitro and Pearson's correlation results were aligned showing promising SARS-CoV-2 inhibitory activity of E. citriodora and globulol. This study is a first record for E. citriodora EO as a novel lead exhibiting potent in vitro, and in silico anti SARS-CoV-2 potential and suggesting its component globulol as a promising candidate for further extensive in silico, in vitro and in vivo anti-COVID studies., (© 2024. The Author(s).)

The main protease (M Pro ) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target M Pro 's catalytic cysteine. Our characterization identified potent M Pro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC 50 value of 10 nM against SARS-CoV-2 infection in ACE2 + A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent M Pro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

COVID-19 is a multisystemic disease caused by the SARS-CoV-2 airborne virus, a member of the Coronaviridae family. It has a positive sense single-stranded RNA genome and encodes two non-structural proteins through viral cysteine-proteases processing. Blocking this step is crucial to control virus replication. In this work, we reported the synthesis of 23 statine-based peptidomimetics to determine their ability to inhibit the main protease (Mpro) activity of SARS-CoV-2. Among the 23 peptidomimetics, 15 compounds effectively inhibited Mpro activity by 50% or more, while three compounds (7d, 8e, and 9g) exhibited maximum inhibition above 70% and IC 50  < 1 µM. Compounds 7d, 8e, and 9g inhibited roughly 80% of SARS-CoV-2 replication and proved no cytotoxicity. Molecular docking simulations show putative hydrogen bond and hydrophobic interactions between specific amino acids and these inhibitors. Molecular dynamics simulations further confirmed the stability and persisting interactions in Mpro's subsites, exhibiting favorable free energy binding (ΔG bind ) values. These findings suggest the statine-based peptidomimetics as potential therapeutic agents against SARS-CoV-2 by targeting Mpro., (© 2024. The Author(s).)

Seven furanochromene-quinoline derivatives containing a hydrazone linker were synthesized by condensing a furanochromene hydrazide with quinoline 2-, 3-, 4-, 5-, 6-, and 8-carbaldehydes, including 8-hydroxyquinoline-2-carbaldehye. Structure-activity correlations were investigated to determine the influence of the location of the hydrazone linker on the quinoline unit on SARS-CoV-2 M pro enzyme inhibition. The 3-, 5-, 6- and 8-substituted derivatives showed moderate inhibition of SARS-CoV-2 M pro with IC 50 values ranging from 16 to 44 μM. Additionally, all of the derivatives showed strong interaction with the SARS-CoV-2 M pro substrate binding pocket, with docking energy scores ranging from -8.0 to -8.5 kcal/mol. These values are comparable to that of N3 peptide (-8.1 kcal/mol) and more favorable than GC-373 (-7.6 kcal/mol) and ML-188 (-7.5 kcal/mol), all of which are known SARS-CoV-2 M pro inhibitors. Furthermore, in silico absorption, distribution, metabolism, and excretion (ADME) profiles indicate that the derivatives have good drug-likeness properties. Overall, this study highlights the potential of the furanochromene-quinoline hydrazone scaffold as a SARS-CoV-2 M pro inhibitor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Small-molecule antivirals that prevent the replication of the SARS-CoV-2 virus by blocking the enzymatic activity of its main protease (Mpro) are and will be a tenet of pandemic preparedness. However, the peptidic nature of such compounds often precludes the design of compounds within favorable physical property ranges, limiting cellular activity. Here we describe the discovery of peptide aldehyde Mpro inhibitors with potent enzymatic and cellular antiviral activity. This structure-activity relationship (SAR) exploration was guided by the use of calculated hydration site thermodynamic maps (WaterMap) to drive potency via displacement of waters from high-energy sites. Thousands of diverse compounds were designed to target these high-energy hydration sites and then prioritized for synthesis by physics- and structure-based Free-Energy Perturbation (FEP+) simulations, which accurately predicted biochemical potencies. This approach ultimately led to the rapid discovery of lead compounds with unique SAR that exhibited potent enzymatic and cellular activity with excellent pan-coronavirus coverage., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: all authors are current or former employees of Takeda Pharmaceuticals, Inc, Schrödinger, Inc, or Treeline Biosciences, Inc. (see affiliations) and may own shares of stock and/or stock options in these companies., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Alongside vaccines and antiviral therapeutics, diagnostic tools are a crucial aid in combating the COVID-19 pandemic caused by the etiological agent SARS-CoV-2. All common assays for infection rely on the detection of viral sub-components, including structural proteins of the virion or fragments of the viral genome. Selective pressure imposed by human intervention of COVID-19 can, however, induce viral mutations that decrease the sensitivity of diagnostic assays based on biomolecular structure, leading to an increase in false-negative results. In comparison, mutations are unlikely to alter the function of viral proteins, and viral machinery is under less selective pressure from vaccines and therapeutics. Accordingly, diagnostic assays that rely on biomolecular function can be more robust than ones that rely on biopolymer structure. Toward this end, we used a split intein to create a circular ribonuclease zymogen that is activated by the SARS-CoV-2 main protease, 3CL pro . Zymogen activation by 3CL pro leads to a >300-fold increase in ribonucleolytic activity, which can be detected with a highly sensitive fluorogenic substrate. This coupled assay can detect low nanomolar concentrations of 3CL pro within a timeframe comparable to that of common antigen-detection protocols. More generally, the concept of detecting a protease by activating a ribonuclease could be the basis of diagnostic tools for other indications., (© 2024 The Protein Society.)

Background: The COVID-19 pandemic began in 2019 as a result of the advent of a novel coronavirus, SARS-CoV-2. At present, there are a limited number of approved antiviral agents for the treatment of COVID-19. Remdesivir, Molnupiravir, and Paxlovid have been approved by the FDA to treat COVID-19 infections. Research has shown that the main protease enzyme (Mpro) of SARS-CoV-2 plays a crucial role in the enzymatic processing of viral polyproteins. This makes Mpro an interesting therapeutic target for combating infections caused by emerging coronaviruses., Methods: The pharmacological effects of pyrroles and their derivatives have a wide range of applications. In our study, we focused on synthesizing nine novel derivatives of 2-arylamino-dihydro-indeno[1,2-b] pyrrol-4(1H)-one, with a particular emphasis on their antiviral properties. Using in silico studies involving molecular docking and DFT analyses in the gas phase using the B3LYP/6-31++G(d,p) basis set, we studied these compounds with respect to their interactions with the Mpro of SARS-CoV-2. The results of the docking analysis revealed that the synthesized compounds exhibited favorable inhibitory effects. Notably, compound 5f demonstrated the highest effectiveness against the target protein. Furthermore, the pharmacokinetic and drug-like properties of the synthesized derivatives of 2-arylamino-dihydroindeno[1,2-b] pyrrol-4(1H)-one indicated their potential as promising candidates for further development as inhibitors targeting SARS-CoV-2. However, it is imperative to determine the in vitro efficacy of these compounds through comprehensive biochemical and structural analyses., Competing Interests: NO authors have competing interests., (Copyright: © 2024 Gheidari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Protein-templated fragment ligation was established as a method for the rapid identification of high affinity ligands, and multicomponent reactions (MCR) such as the Ugi four-component reaction (Ugi 4CR) have been efficient in the synthesis of drug candidates. Thus, the combination of both strategies should provide a powerful approach to drug discovery. Here, we investigate protein-templated Ugi 4CR quantitatively using a fluorescence-based enzyme assay, HPLC-QTOF mass spectrometry (MS), and native protein MS with SARS-CoV-2 main protease as template. Ugi reactions were analyzed in aqueous buffer at varying pH and fragment concentration. Potent inhibitors of the protease were formed in presence of the protein via Ugi 4CR together with Ugi three-component reaction (Ugi 3CR) products. Binding of inhibitors to the protease was confirmed by native MS and resulted in the dimerization of the protein target. Formation of Ugi products was, however, more efficient in the non-templated reaction, apparently due to interactions of the protein with the isocyanide and imine fragments. Consequently, in-situ ligation screening of Ugi 4CR products was identified as a superior approach to the discovery of SARS-CoV-2 protease inhibitors., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

To date, Proteolysis Targeting Chimera (PROTAC) technology has been successfully applied to mediate proteasomal-induced degradation of several pharmaceutical targets mainly related to oncology, immune disorders, and neurodegenerative diseases. On the other hand, its exploitation in the field of antiviral drug discovery is still in its infancy. Recently, we described two indomethacin (INM)-based PROTACs displaying broad-spectrum antiviral activity against coronaviruses. Here, we report the design, synthesis, and characterization of a novel series of INM-based PROTACs that recruit either Von-Hippel Lindau (VHL) or cereblon (CRBN) E3 ligases. The panel of INM-based PROTACs was also enlarged by varying the linker moiety. The antiviral activity resulted very susceptible to this modification, particularly for PROTACs hijacking VHL as E3 ligase, with one piperazine-based compound (PROTAC 6) showing potent anti-SARS-CoV-2 activity in infected human lung cells. Interestingly, degradation assays in both uninfected and virus-infected cells with the most promising PROTACs emerged so far (PROTACs 5 and 6) demonstrated that INM-PROTACs do not degrade human PGES-2 protein, as initially hypothesized, but induce the concentration-dependent degradation of SARS-CoV-2 main protease (M pro ) both in M pro -transfected and in SARS-CoV-2-infected cells. Importantly, thanks to the target degradation, INM-PROTACs exhibited a considerable enhancement in antiviral activity with respect to indomethacin, with EC 50 values in the low-micromolar/nanomolar range. Finally, kinetic solubility as well as metabolic and chemical stability were measured for PROTACs 5 and 6. Altogether, the identification of INM-based PROTACs as the first class of SARS-CoV-2 M pro degraders demonstrating activity also in SARS-CoV-2-infected cells represents a significant advance in the development of effective, broad-spectrum anti-coronavirus strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

A novel in silico drug design procedure is described targeting the Main protease (Mpro) of the SARS-CoV-2 virus. The procedure combines molecular docking, molecular dynamics (MD), and fragment molecular orbital (FMO) calculations. The binding structure and properties of Mpro were predicted for Nelfinavir (NFV), which had been identified as a candidate compound through drug repositioning, targeting Mpro. Several poses of the Mpro and NFV complexes were generated by docking, from which four docking poses were selected by scoring with FMO energy. Then, each pose was subjected to MD simulation, 100 snapshot structures were sampled from each of the generated MD trajectories, and the structures were evaluated by FMO calculations to rank the pose based on binding energy. Several residues were found to be important in ligand recognition, including Glu47, Asp48, Glu166, Asp187, and Gln189, all of which interacted strongly with NFV. Asn142 is presumably regarded to form hydrogen bonds or CH/π interaction with NFV; however, in the present calculation, their interactions were transient. Moreover, the tert -butyl group of NFV had no interaction with Mpro. Identifying such strong and weak interactions provides candidates for maintaining and substituting ligand functional groups and important suggestions for drug discovery using drug repositioning. Besides the interaction between NFV and the amino acid residues of Mpro, the desolvation effect of the binding pocket also affected the ranking order. A similar procedure of drug design was applied to Lopinavir, and the calculated interaction energy and experimental inhibitory activity value trends were consistent. Our approach provides a new guideline for structure-based drug design starting from a candidate compound whose complex crystal structure has not been obtained.

Inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M pro ) such as nirmatrelvir (NTV) and ensitrelvir (ETV) have proven effective in reducing the severity of COVID-19, but the presence of resistance-conferring mutations in sequenced viral genomes raises concerns about future drug resistance. Second-generation oral drugs that retain function against these mutants are thus urgently needed. We hypothesized that the covalent hepatitis C virus protease inhibitor boceprevir (BPV) could serve as the basis for orally bioavailable drugs that inhibit SARS-CoV-2 M pro more efficiently than existing drugs. Performing structure-guided modifications of BPV, we developed a picomolar-affinity inhibitor, ML2006a4, with antiviral activity, oral pharmacokinetics, and therapeutic efficacy similar or superior to those of NTV. A crucial feature of ML2006a4 is a derivatization of the ketoamide reactive group that improves cell permeability and oral bioavailability. Last, ML2006a4 was found to be less sensitive to several mutations that cause resistance to NTV or ETV and occur in the natural SARS-CoV-2 population. Thus, anticipatory design can preemptively address potential resistance mechanisms to expand future treatment options against coronavirus variants.

The SARS-CoV-2 virus and its mutations have affected human health globally and created significant danger for the health of people all around the world. To cure this virus, the human Angiotensin Converting Enzyme-2 (ACE2) receptor, the SARS-CoV-2 main protease (Mpro), and spike proteins were found to be likely candidates for the synthesis of novel therapeutic drug. In the past, proteins were capable of engaging in interaction with a wide variety of ligands, including both manmade and plant-derived small molecules. Pyrus communis L., Ginko bibola , Carica papaya , Syrian rue , and Pimenta dioica were some of the plant species that were studied for their tendency to interact with SARS-CoV-2 main protease (Mpro) in this research project (6LU7). This scenario investigates the geometry, electronic, and thermodynamic properties computationally. Assessing the intermolecular forces of phytochemicals with the targets of the SARS-CoV-2 Mpro spike protein (SP) resulted in the recognition of a compound, kaempferol, as the most potent binding ligand, -7.7 kcal mol -1 . Kaempferol interacted with ASP-187, CYS-145, SER-144, LEU 141, MET-165, and GLU-166 residues. Through additional molecular dynamic simulations, the stability of ligand-protein interactions was assessed for 100 ns. GLU-166 remained intact with 33% contact strength with phenolic OH group. We noted a change in torsional conformation, and the molecular dynamics simulation showed a potential variation in the range from 3.36 to 7.44 against a 45-50-degree angle rotation. SAR, pharmacokinetics, and drug-likeness characteristic investigations showed that kaempferol may be the suitable candidate to serve as a model for designing and developing new anti-COVID-19 medicines.

The outbreak of coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health. In parallel with vaccines, efficacious antivirals are urgently needed. SARS-CoV-2 main protease (Mpro) is an attractive drug target for antiviral development owing to its key roles in virus replication and host immune evasion. Due to the limitations of currently available methods, the development of novel high-throughput screening assays is of the highest importance for the discovery of Mpro inhibitors. In this study, we first developed an improved fluorescence-based assay for rapid screening of Mpro inhibitors from an anti-infection compound library using a versatile dimerization-dependent red fluorescent protein (ddRFP) biosensor. Utilizing this assay, we identified MG-101 as a competitive Mpro inhibitor in vitro. Moreover, our results revealed that ensitrelvir is a potent Mpro inhibitor, but baicalein, chloroquine, ebselen, echinatin, and silibinin are not. Therefore, this robust ddRFP assay provides a faithful avenue for rapid screening and evaluation of Mpro inhibitors to fight against COVID-19., (© 2024 Wiley Periodicals LLC.)

The main protease (M pro ) enzyme has an imperative function in disease progression and the life cycle of the SARS-CoV-2 virus. Although the orally active drug nirmatrelvir (co-administered with ritonavir as paxlovid) has been approved for emergency use as the frontline antiviral agent, there are a number of limitations that necessitate the discovery of new drug scaffolds, such as poor pharmacokinetics and susceptibility to proteolytic degradation due to its peptidomimetic nature. This study utilized a novel virtual screening workflow that combines pharmacophore modelling, multiple-receptor covalent docking, and biological evaluation in order to find new M pro inhibitors. After filtering and analysing ∼66,000 ligands from three different electrophilic libraries, 29 compounds were shortlisted for experimental testing, and two of them exhibited ≥20% inhibition at 100 μM. Our top candidate, GF04, is a benzylpyrrolyl compound that exhibited the highest inhibition activity of 38.3%, with a relatively small size (<350 Da) and leadlike character. Interestingly, our approach also identified another hit, DR07, a pyrimidoindol with a non-peptide character, and a molecular weight of 438.9 Da, reporting an inhibition of 26.3%. The established approach detailed in this study, in conjunction with the discovered inhibitors, has the capacity to yield novel perspectives for devising covalent inhibitors targeting the COVID-19 M pro enzyme and other comparable targets., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Mohammad A. Ghattas reports financial support was provided by Al Jalila Foundation., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Cranberry phytochemicals are known to possess antiviral activities. In the current study, we explored the therapeutic potential of cranberry against SARS-CoV-2 by targeting its main protease (M pro ) enzyme. Firstly, phytochemicals of cranberry origin were identified from three independent databases. Subsequently, virtual screening, using molecular docking and molecular dynamics simulation approaches, led to the identification of three lead phytochemicals namely, cyanidin 3-O-galactoside, β-carotene and epicatechin. Furthermore, in vitro enzymatic assays revealed that cyanidin 3-O-galactoside had the highest inhibitory potential with IC 50 of 9.98 μM compared to the other two phytochemicals. Cyanidin 3-O-galactoside belongs to the class of anthocyanins. Anthocyanins extracted from frozen cranberry also exhibited the highest inhibitory potential with IC 50 of 23.58 μg/ml compared to the extracts of carotenoids and flavanols, the class for β-carotene and epicatechin, respectively. Finally, we confirm the presence of the phytochemicals in the cranberry extracts using targeted LC-MS/MS analysis. Our results, therefore, indicate that the identified cranberry-derived bioactive compounds as well as cranberry could be used for therapeutic interventions against SARS-CoV-2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Traditional Chinese medicine (TCM) has been extensively employed for the treatment of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there is demand for discovering more SARS-CoV-2 Mpro inhibitors with diverse scaffolds to optimize anti-SARS-CoV-2 lead compounds. In this study, comprehensive in silico and in vitro assays were utilized to determine the potential inhibitors from TCM compounds against SARS-CoV-2 Mpro, which is an important therapeutic target for SARS-CoV-2. The ensemble docking analysis of 18263 TCM compounds against 15 SARS-CoV-2 Mpro conformations identified 19 TCM compounds as promising candidates. Further in vitro testing validated three compounds as inhibitors of SARS-CoV-2 Mpro and showed IC 50 values of 4.64 ± 0.11, 7.56 ± 0.78, and 11.16 ± 0.26 μM, with EC 50 values of 12.25 ± 1.68, 15.58 ± 0.77, and 29.32 ± 1.25 μM, respectively. Molecular dynamics (MD) simulations indicated that the three complexes remained stable over the last 100 ns of production run. An analysis of the binding mode revealed that the active compounds occupy different subsites (S1, S2, S3, and S4) of the active site of SARS-CoV-2 Mpro via specific poses through noncovalent interactions with key amino acids (e.g., HIS 41, ASN 142, GLY 143, MET 165, GLU 166, or GLN 189). Overall, this study provides evidence indicating that the three natural products obtained from TCM could be further used for anti-COVID-19 research, justifying the investigation of Chinese herbal medicinal ingredients as bioactive constituents for therapeutic targets.

The conventional peptide substrates of SARS-CoV-2 main protease (Mpro) are frequently associated with high cost, unstable kinetics, and multistep synthesis. Hence, there is an urgent need to design affordable and stable Mpro substrates for pharmacological research. Herein, we designed a functional Mpro substrate based on a dimerization-dependent red fluorescent protein (ddRFP) for the evaluation of Mpro inhibitors in vitro . The codon-optimized DNA fragment encoding RFP-A 1 domain, a polypeptide linker containing Mpro cleavage sequence (AVLQS), and the RFP-B 1 domain was subcloned into the pET-28a vector. After transformation into Escherichia coli Rosetta(DE3) cells, the kanamycin resistant transformants were selected. Using a low temperature induction strategy, most of the target proteins (ddRFP-M) presented in the supernatant fractions were collected and purified by a HisTrap TM chelating column. Subsequently, the inhibition of Mpro by ensitrelvir and baicalein was assessed using ddRFP-M assay, and the biochemical properties of ddRFP-M substrate were analyzed. Our results showed that the fluorogenic substrate ddRFP-M was successfully prepared from E . coli cells, and this biosensor exhibited the expected specificity, sensitivity, and reliability. In conclusion, the production of the fluorogenic substrate ddRFP-M provides an expedient avenue for the assessment of Mpro inhibitors in vitro .

Highly contagious SARS-CoV-2 coronavirus has infected billions of people worldwide with flu-like symptoms since its emergence in 2019. It has caused deaths of several million people. The viral main protease (Mpro) is essential for SARS-CoV-2 replication and therefore a drug target. Several series of covalent inhibitors of Mpro were designed and synthesized. Structure-activity relationship studies show that (1) several chloroacetamide- and epoxide-based compounds targeting Cys145 are potent inhibitors with IC 50 values as low as 0.49 μM and (2) Cys44 of Mpro is not nucleophilic for covalent inhibitor design. High-resolution X-ray studies revealed the protein-inhibitor interactions and mechanisms of inhibition. It is of interest that Cys145 preferably attacks the more hindered C α atom of several epoxide inhibitors. Chloroacetamide inhibitor 13 and epoxide inhibitor 30 were found to inhibit cellular SARS-CoV-2 replication with an EC 68 (half-log reduction of virus titer) of 3 and 5 μM. These compounds represent new pharmacological leads for anti-SARS-CoV-2 drug development.

Although the SARS-CoV-2 pandemic has ended, multiple sporadic cases still exist, posing a request for more antivirals. The main protease (M pro ) of SARS-CoV-2, a key enzyme for viral replication, is an attractive target for drug development. Here, we report the discovery of a new potent α-ketoamide-containing M pro inhibitor, N-((R)-1-cyclohexyl-2-(((R)-3-methoxy-1-oxo-1-((1-(2-oxo-2-((thiazol-2-ylmethyl)amino)acetyl)cyclobutyl)amino)propan-2-yl)amino)-2-oxoethyl)-4,4-difluorocyclohexane-1-carboxamide (20j). This compound presented promising enzymatic inhibitory activity against SARS-CoV-2 M pro with an IC 50 value of 19.0 nM, and an excellent antiviral activity in cell-based assay with an EC 50 value of 138.1 nM. This novel covalent inhibitor may be used as a lead compound for subsequent drug discovery against SARS-CoV-2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Viral infections cause significant health problems all over the world, and it is critical to develop treatments for these problems. Antivirals that target viral genome-encoded proteins frequently cause the virus to become more resistant to treatment. Because viruses rely on several cellular proteins and phosphorylation processes that are essential to their life cycle, drugs targeting host-based targets could be a viable treatment option. To reduce costs and improve efficiency, existing kinase inhibitors could be repurposed as antiviral medications; however, this method rarely works, and specific biophysical approaches are required in the field. Because of the widespread use of FDA-approved kinase inhibitors, it is now possible to better understand how host kinases contribute to viral infection. The purpose of this article is to investigate the tyrphostin AG879 (Tyrosine kinase inhibitor) binding information in Bovine Serum Albumin (BSA), human ErbB2 (HER2), C-RAF1 Kinase (c-RAF), SARS-CoV-2 main protease (COVID 19), and Angiotensin-converting enzyme 2 (ACE-2).Communicated by Ramaswamy H. Sarma.

To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography., (open access.)

COVID-19, the disease responsible for the recent pandemic, is caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (Mpro) of SARS-CoV-2 is an essential proteolytic enzyme that plays a number of important roles in the replication of the virus in human host cells. Blocking the function of SARS-CoV-2 Mpro offers a promising and targeted, therapeutic option for the treatment of the COVID-19 infection. Such an inhibitory strategy is currently successful in treating COVID-19 under FDA's emergency use authorization, although with limited benefit to the immunocompromised along with an unfortunate number of side effects and drug-drug interactions. Current COVID vaccines protect against severe disease and death but are mostly ineffective toward long COVID which has been seen in 5-36% of patients. SARS-CoV-2 is a rapidly mutating virus and is here to stay endemically. Hence, alternate therapeutics to treat SARS-CoV-2 infections are still needed. Moreover, because of the high degree of conservation of Mpro among different coronaviruses, any newly developed antiviral agents should better prepare us for potential future epidemics or pandemics. In this paper, we first describe the design and computational docking of a library of novel 188 first-generation peptidomimetic protease inhibitors using various electrophilic warheads with aza-peptide epoxides, α-ketoesters, and β-diketones identified as the most effective. Second-generation designs, 192 compounds in total, focused on aza-peptide epoxides with drug-like properties, incorporating dipeptidyl backbones and heterocyclic ring motifs such as proline, indole, and pyrrole groups, yielding 8 hit candidates. These novel and specific inhibitors for SARS-CoV-2 Mpro can ultimately serve as valuable alternate and broad-spectrum antivirals against COVID-19.Communicated by Ramaswamy H. Sarma.

The implementation of innovative approaches is crucial in an ongoing endeavor to mitigate the impact of COVID-19 pandemic. The present study examines the strategic application of the SARS-CoV-2 Main Protease (M pro ) as a prospective instrument in the repertoire to combat the virus. The cloning, expression, and purification of M pro , which plays a critical role in the viral life cycle, through heterologous expression in Escherichia coli in a completely soluble form produced an active enzyme. The hydrolysis of a specific substrate peptide comprising a six-amino-acid sequence (TSAVLQ) linked to a p-nitroaniline (pNA) fragment together with the use of a fluorogenic substrate allowed us to determine effective inhibitors incorporating selenium moieties, such as benzoselenoates and carbamoselenoates. The new inhibitors revealed their potential to proficiently inhibit M pro with IC 50 -s in the low micromolar range. Our study contributes to the development of a new class of protease inhibitors targeting M pro , ultimately strengthening the antiviral arsenal against COVID-19 and possibly, related coronaviruses.