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The persistence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new mutant strains continue to present a substantial threat with potential for future pandemics. Safe, effective, and readily available COVID-19 therapeutics are urgently needed to prepare for future coronavirus pandemics. To help identify new antiviral agents, the present study focused on natural products in the extracts of Holy Basil, Ocimum tenuiflorum L., which show potential inhibitory effects against the SARS-CoV-2 main protease (M pro ). Bioassay-guided isolation of the MeOH extracts of O. tenuiflorum led to the identification of a sulfur-containing glyceroglycolipid, sulfoquinovosyl diacylglycerol (SQDG: 1), as a potent M pro inhibitor that effectively inhibited M pro activity (IC 50 : 0.42 µM). SQDG (1) also markedly suppressed SARS-CoV-2 replication (EC 50 , 51.2 µM) in vitro while displaying no cytotoxicity (CC 50 > 100 µM). Further inhibition kinetic studies and docking simulations clearly demonstrated that SQDG strongly inhibited SARS-CoV-2 M pro in a competitive and mixed-inhibition manner. These findings highlight SQDG as a promising lead compound for COVID-19 therapy and emphasize the need to explore new drugs from natural sources., Competing Interests: Declarations. Conflict of interest: The authors declare no competing financial interests., (© 2024. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M pro ) autocatalytically releases itself out of the viral polyprotein to form a fully active mature dimer in a manner that is not fully understood. Here, we introduce several tools to help elucidate differences between cis (intramolecular) and trans (intermolecular) proteolytic processing and to evaluate inhibition of precursor M pro . We found that many mutations at the P1 position of the N-terminal autoprocessing site do not block cis autoprocessing but do inhibit trans processing. Notably, substituting the WT glutamine at the P1 position with isoleucine retains M pro in an unprocessed precursor form that can be purified and further studied. We also developed a cell-based reporter assay suitable for compound library screening and evaluation in HEK293T cells. This assay can detect both overall M pro inhibition and the fraction of uncleaved precursor form of M pro through separable fluorescent signals. We observed that inhibitory compounds preferentially block mature M pro . Bofutrelvir and a novel compound designed in-house showed the lowest selectivity between precursor and mature M pro , indicating that inhibition of both forms may be possible. Additionally, we observed positive modulation of precursor activity at low concentrations of inhibitors. Our findings help expand understanding of the SARS-CoV-2 viral life cycle and may facilitate development of strategies to target precursor form of M pro for inhibition or premature activation of M pro ., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We investigate the inhibition mechanism between pomotrelvir and the SARS-CoV-2 main protease using molecular mechanics and quantum mechanics/molecular mechanics simulations. Alchemical transformations where each Pi group of pomotrelvir was transformed into its counterpart in nirmatrelvir were performed to unravel the individual contribution of each group to the binding and reaction processes. We have shown that while a γ-lactam ring is preferred at position P1, a δ-lactam ring is a good alternative for the design of inhibitors for variants presenting mutations at position 166. For the P2 position, tertiary amines are preferred with respect to secondary amines. Flexible side chains at the P2 position can disrupt the preorganization of the active site, favouring the exploration of non-reactive conformations. The substitution of the P2 group of pomotrelvir by that of nirmatrelvir resulted in a compound, named as C2, that presents a better binding free energy and a higher population of reactive conformations in the Michaelis complex. Analysis of the chemical reaction to form the covalent complex has shown a similar reaction mechanism and activation free energies for pomotrelvir, nirmatrelvir and C2. We hope that these findings could be useful to design better inhibitors to fight present and future variants of the SARS-CoV-2 virus., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The global outbreak of the COVID-19 pandemic caused by the SARS-CoV-2 virus had led to profound respiratory health implications. This study focused on designing organoselenium-based inhibitors targeting the SARS-CoV-2 main protease (M pro ). The ligand-binding pathway sampling method based on parallel cascade selection molecular dynamics (LB-PaCS-MD) simulations was employed to elucidate plausible paths and conformations of ebselen, a synthetic organoselenium drug, within the M pro catalytic site. Ebselen effectively engaged the active site, adopting proximity to H41 and interacting through the benzoisoselenazole ring in a π-π T-shaped arrangement, with an additional π-sulfur interaction with C145. In addition, the ligand-based drug design using the QSAR with GFA-MLR, RF, and ANN models were employed for biological activity prediction. The QSAR-ANN model showed robust statistical performance, with an r 2 training exceeding 0.98 and an RMSE test of 0.21, indicating its suitability for predicting biological activities. Integration the ANN model with the LB-PaCS-MD insights enabled the rational design of novel compounds anchored in the ebselen core structure, identifying promising candidates with favorable predicted IC 50 values. The designed compounds exhibited suitable drug-like characteristics and adopted an active conformation similar to ebselen, inhibiting M pro function. These findings represent a synergistic approach merging ligand and structure-based drug design; with the potential to guide experimental synthesis and enzyme assay testing., 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.)

The assembly of two monomeric constructs spanning segments 1-199 (MPro 1-199 ) and 10-306 (MPro 10-306 ) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ∼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro 1-199 and MPro 10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro 10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro 1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro 10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ∼6 μM MPro 1-199 . Catalytic activity arises primarily from MPro 1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro 1-199 and E290 of MPro 10-306 . Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

SARS-CoV-2 main protease (Mpro) is a well-recognized target for COVID-19 therapy. Green tea (-)-epigallocatechin-3-gallate (EGCG) possesses Mpro-inhibitory activity; however, the influence of EGCG oxidation on its inhibition activity remains obscure, given its high oxidation propensity. This study reveals that prolonged EGCG oxidation in the presence of Mpro dramatically increases its inhibitory activity with an IC 50 of 0.26 μM. The inhibitory mechanism is that EGCG-quinone preferentially binds the active site Mpro-Cys145-SH, which forms a quinoprotein. Though Mpro is present in the cell lysate, EGCG preferentially depletes its thiols. Non-cytotoxic EGCG effectively generates a quinoprotein in living cells, thus EGCG might selectively inhibit Mpro in SARS-CoV-2 infected cells. Chlorogenic acid facilitates EGCG oxidation. Together, they synergistically deplete multiple Mpro thiols though this is not more beneficial than EGCG alone. By contrast, excessive EGCG oxidation prior to incubation with Mpro largely compromises its inhibitory activity. Overall, the low IC 50 and the high selectivity imply that EGCG is a promising dietary Mpro inhibitor. While EGCG oxidation in the presence of Mpro has a pivotal role in inhibition, enhancing EGCG oxidation by chlorogenic acid no longer increases its inhibitory potential. EGCG oxidation in the absence of Mpro should be avoided to maximize its Mpro-inhibitory activity., 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.)

The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (M pro ) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a K i value of 48 ± 5 μM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.

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.

The Coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) has become a global health crisis, and the urgent need for effective treatments is evident. One potential target for COVID-19 therapeutics is the main protease (Mpro) of SARS‑CoV‑2, an essential enzyme for viral replication. Natural compounds have been explored as a source of potential inhibitors for Mpro due to their safety and availability. In this study, we employed a computational approach to screen a library of phytoconstituents and identified potential Mpro inhibitors based on their binding affinities and molecular interactions. The top-ranking compounds were further validated through molecular dynamics simulations (MDS) and free energy calculations. As a result of the above procedures, we identified two phytoconstituents, Khelmarin B and Neogitogenin, with appreciable binding affinity and specificity towards the Mpro binding pocket. Our results suggest that Khelmarin B and Neogitogenin could potentially serve as Mpro inhibitors and have the potential to be developed as COVID-19 therapeutics. Further experimental studies are required to confirm the efficacy and safety of these compounds.Communicated by Ramaswamy H. Sarma.

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

The SARS-CoV-2 main protease (M pro ) is responsible for cleaving twelve nonstructural proteins from the viral polyprotein. M pro , a cysteine protease, is characterized by a large number of noncatalytic cysteine (Cys) residues, none involved in disulfide bonds. In the absence of a tertiary-structure stabilizing role for these residues, a possible alternative is that they are involved in redox processes. We report experimental work in support of a proposal that surface cysteines on M pro can protect the active-site Cys145 from oxidation by reactive oxygen species (ROS). In investigations of enzyme kinetics, we found that mutating three surface cysteines to serines did not greatly affect activity, which in turn indicates that these cysteines could protect Cys145 from oxidative damage., (Copyright © 2022 Elsevier Inc. All rights reserved.)

SignificanceThe coronavirus main protease (M pro ) is required for viral replication. Here, we obtained the extended conformation of the native monomer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M pro by trapping it with nanobodies and found that the catalytic domain and the helix domain dissociate, revealing allosteric targets. Another monomeric state is termed compact conformation and is similar to one protomer of the dimeric form. We designed a Nanoluc Binary Techonology (NanoBiT)-based high-throughput allosteric inhibitor assay based on structural conformational change. Our results provide insight into the maturation, dimerization, and catalysis of the coronavirus M pro and pave a way to develop an anticoronaviral drug through targeting the maturation process to inhibit the autocleavage of M pro .

Since the time of its appearance until present, COVID-19 has spread worldwide, with over 71 million confirmed cases and over 1.6 million deaths reported by the World Health Organization (WHO). In addition to the fact that cases of COVID-19 are increasing worldwide, the Delta and Omicron variants have also made the situation more challenging. Herein, we report the evaluation of several thiazole/thiadiazole/benzothiazole based thiazolidinone derivatives which were chosen from 112 designed derivatives by docking as potential molecules to inhibit the main protease of SARS-CoV-2. The contained experimental data revealed that among the fifteen compounds chosen, five compounds ( k3 , c1 , n2 , A2 , A1 ) showed inhibitory activity with IC 50 within the range of 0.01-34.4 μΜ. By assessing the cellular effects of these molecules, we observed that they also had the capacity to affect the cellular viability of human normal MRC-5 cells, albeit with a degree of variation. More specifically, k3 which is the most promising compound with the higher inhibitory capacity to SARS-CoV-2 protease (0.01 μΜ) affects in vitro cellular viability only by 57% at the concentration of 0.01 μM after 48 h in culture. Overall, these data provide evidence on the potential antiviral activity of these molecules to inhibit the main protease of SARS-CoV-2, a fact that sheds light on the chemical structure of the thiazole/thiadiazole/benzothiazole based thiazolidin-4-one derivatives as potential candidates for COVID-19 therapeutics.

One of the emerging subjects to combat the SARS-CoV-2 virus is to design accurate and efficient drug such as inhibitors against the viral protease to stop the viral spread. In addition to laboratory investigation of the viral protease, which is fundamental, the in silico research of viral protease such as the protease cleavage site prediction is critically important and urgent. However, this problem has yet to be addressed. This article has, for the first time, investigated this problem using the pattern recognition approaches. The article has shown that the pattern recognition approaches incorporating a specially tailored kernel function for dealing with amino acids has the outstanding performance in the accuracy of cleavage site prediction and the discovery of the prototype cleavage peptides., (© 2021 Wiley Periodicals LLC.)

The COVID-19 new variants spread rapidly all over the world, and until now scientists strive to find virus-specific antivirals for its treatment. The main protease of SARS-CoV-2 (M pro ) exhibits high structural and sequence homology to main protease of SARS-CoV (93.23% sequence identity), and their sequence alignment indicated 12 mutated/variant residues. The sequence alignment of SARS-CoV-2 main protease led to identification of only one mutated/variant residue with no significant role in its enzymatic process. Therefore, M pro was considered as a high-profile drug target in anti-SARS-CoV-2 drug discovery. Apigenin analogues to COVID-19 main protease binding were evaluated. The detailed interactions between the analogues of Apigenin and SARS-CoV-2 M pro inhibitors were determined as hydrogen bonds, electronic bonds and hydrophobic interactions. The binding energies obtained from the molecular docking of M pro with Boceprevir, Apigenin, Apigenin 7-glucoside-4'-p-coumarate, Apigenin 7-glucoside-4'-trans-caffeate and Apigenin 7-O-beta-d-glucoside (Cosmosiin) were found to be -6.6, -7.2, -8.8, -8.7 and -8.0 kcal/mol, respectively. Pharmacokinetic parameters and toxicological characteristics obtained by computational techniques and Virtual ADME studies of the Apigenin analogues confirmed that the Apigenin 7-glucoside-4'-p-coumarate is the best candidate for SARS-CoV-2 M pro inhibition.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) has been regarded as one of the ideal targets for the development of antiviral drugs. The currently used methods for the probing of Mpro activity and the screening of its inhibitors require the use of a double-labeled peptide substrate. In this work, we suggested that the label-free peptide substrate could induce the aggregation of AuNPs through the electrostatic interactions, and the cleavage of the peptide by the Mpro inhibited the aggregation of AuNPs. This fact allowed for the visual analysis of Mpro activity by observing the color change of the AuNPs suspension. Furthermore, the co-assembly of AuNPs and peptide was achieved on the peptide-covered electrode surface. Cleavage of the peptide substrate by the Mpro limited the formation of AuNPs/peptide assembles, thus allowing for the development of a simple and sensitive electrochemical method for Mpro detection in serum samples. The change of the electrochemical signal was easily monitored by electrochemical impedance spectroscopy (EIS). The detection limits of the colorimetric and electrochemical methods are 10 and 0.1 pM, respectively. This work should be valuable for the development of effective antiviral drugs and the design of novel optical and electrical biosensors.

The main protease (M pro ) of SARS-CoV-2 is responsible for the cleavage of viral replicase polyproteins 1a and 1ab into their mature form and is highly specific and exclusive in its activity. Many studies have targeted this enzyme by small molecule inhibitors to develop therapeutics against the highly infectious disease Covid-19. Our diet contains many natural antioxidants which along with providing support for proper growth and functioning of the body, pose additional health benefits. Present in-silico analysis depicted that natural antioxidants like sesamin, ellagic acid, capsaisin, and epicatechin along with galangin, exhibited significant binding at the catalytic site of the M pro enzyme. They interacted with excellent efficiency with the chief active site residue Cys145 and thus seem to possess the remarkable potential to act as drug candidates for the treatment of Covid-19. Such dietary compounds can be easily administered orally with least toxicity related concern and thus yell for urgent exhaustive research to develop into efficient therapies.Communicated by Ramaswamy H. Sarma.

The main viral protease (3CL pro ) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CL pro by TAILS substrate-targeted N-terminomics. We identify more than 100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CL pro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CL pro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral-autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike in healthy lungs. The 3CL pro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The inhibition mechanism of the main protease (M pro ) of SARS-CoV-2 by ebselen (EBS) and its analog with a hydroxyl group at position 2 of the benzisoselenazol-3(2H)-one ring (EBS-OH) was studied by using a density functional level of theory. Preliminary molecular dynamics simulations on the apo form of M pro were performed taking into account both the hydrogen donor and acceptor natures of the Nδ and Nε of His41, a member of the catalytic dyad. The potential energy surfaces for the formation of the Se-S covalent bond mediated by EBS and EBS-OH on M pro are discussed in detail. The EBS-OH shows a distinctive behavior with respect to EBS in the formation of the noncovalent complex. Due to the presence of canonical H-bonds and noncanonical ones involving less electronegative atoms, such as sulfur and selenium, the influence on the energy barriers and reaction energy of the Minnesota hybrid meta-GGA functionals M06, M06-2X and M08HX, and the more recent range-separated hybrid functional wB97X were also considered. The knowledge of the inhibition mechanism of M pro by the small protease inhibitors EBS or EBS-OH can enlarge the possibilities for designing more potent and selective inhibitor-based drugs to be used in combination with other antiviral therapies.

The emergence and rapid spreading of novel SARS-CoV-2 across the globe represent an imminent threat to public health. Novel antiviral therapies are urgently needed to overcome this pandemic. Given the significant role of the main protease of Covid-19 for virus replication, we performed a drug-repurposing study using the recently deposited main protease structure, 6LU7. For instance, pharmacophore- and e-pharmacophore-based hypotheses such as AARRH and AARR, respectively, were developed using available small molecule inhibitors and utilized in the screening of the DrugBank repository. Further, a hierarchical docking protocol was implemented with the support of the Glide algorithm. The resultant compounds were then examined for their binding free energy against the main protease of Covid-19 by means of the Prime-MM/GBSA algorithm. Most importantly, the machine learning-based AutoQSAR algorithm was used to predict the antiviral activities of resultant compounds. The hit molecules were also examined for their drug-likeness and toxicity parameters through the QikProp algorithm. Finally, the hit compounds activity against the main protease was validated using molecular dynamics simulation studies. Overall, the present analysis yielded two potential inhibitors (DB02986 and DB08573) that are predicted to bind with the main protease of Covid-19 better than currently used drug molecules such as N3 (cocrystallized native ligand), lopinavir, and ritonavir., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

The coronavirus pandemic has affected more than 150 million people, while over 3.25 million people have died from the coronavirus disease 2019 (COVID-19). As there are no established therapies for COVID-19 treatment, drugs that inhibit viral replication are a promising target; specifically, the main protease (M pro ) that process CoV-encoded polyproteins serves as an Achilles heel for assembly of replication-transcription machinery as well as down-stream viral replication. In the search for potential antiviral drugs that target M pro , a series of cembranoid diterpenes from the biologically active soft-coral genus Sarcophyton have been examined as SARS-CoV-2 M pro inhibitors. Over 360 metabolites from the genus were screened using molecular docking calculations. Promising diterpenes were further characterized by molecular dynamics (MD) simulations based on molecular mechanics-generalized Born surface area (MM-GBSA) binding energy calculations. According to in silico calculations, five cembranoid diterpenes manifested adequate binding affinities as M pro inhibitors with Δ G binding < -33.0 kcal/mol. Binding energy and structural analyses of the most potent Sarcophyton inhibitor, bislatumlide A ( 340 ), was compared to darunavir, an HIV protease inhibitor that has been recently subjected to clinical-trial as an anti-COVID-19 drug. In silico analysis indicates that 340 has a higher binding affinity against M pro than darunavir with Δ G binding values of -43.8 and -34.8 kcal/mol, respectively throughout 100 ns MD simulations. Drug-likeness calculations revealed robust bioavailability and protein-protein interactions were identified for 340 ; biochemical signaling genes included ACE, MAPK14 and ESR1 as identified based on a STRING database. Pathway enrichment analysis combined with reactome mining revealed that 340 has the capability to re-modulate the p38 MAPK pathway hijacked by SARS-CoV-2 and antagonize injurious effects. These findings justify further in vivo and in vitro testing of 340 as an antiviral agent against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emanating human infectious coronavirus that causes COVID-19 disease. On 11th March 2020, it has been announced as a pandemic by the World Health Organization (WHO). Recently, several repositioned drugs have been subjected to clinical investigations as anti-COVID-19 drugs. Here, in silico drug discovery tools were utilized to evaluate the binding affinities and features of eighteen anti-COVID-19 drug candidates against SARS-CoV-2 main protease (M pro ). Molecular docking calculations using Autodock Vina showed considerable binding affinities of the investigated drugs with docking scores ranging from - 5.3 to - 8.3 kcal/mol, with higher binding affinities for HIV drugs compared to the other antiviral drugs. Molecular dynamics (MD) simulations were performed for the predicted drug-M pro complexes for 50 ns, followed by binding energy calculations utilizing molecular mechanics-generalized Born surface area (MM-GBSA) approach. MM-GBSA calculations demonstrated promising binding affinities of TMC-310911 and ritonavir towards SARS-CoV-2 M pro , with binding energy values of - 52.8 and - 49.4 kcal/mol, respectively. Surpass potentialities of TMC-310911 and ritonavir are returned to their capabilities of forming multiple hydrogen bonds with the proximal amino acids inside M pro 's binding site. Structural and energetic analyses involving root-mean-square deviation, binding energy per-frame, center-of-mass distance, and hydrogen bond length demonstrated the stability of TMC-310911 and ritonavir inside the M pro 's active site over the 50 ns MD simulation. This study sheds light on HIV protease drugs as prospective SARS-CoV-2 M pro inhibitors.

The main protease of SARS-CoV-2 (M pro ), the causative agent of COVID-19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed M pro . Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in-depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of M pro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards M pro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (k inac /K i =37 500 m -1  s -1 , K i =24.0 nm) and pyridyl ester 17 (k inac /K i =29 100 m -1  s -1 , K i =10.0 nm), promising drug candidates for further development have been discovered., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the COVID-19 pandemic is ongoing, with no proven safe and effective vaccine to date. Further, effective therapeutic agents for COVID-19 are limited, and as a result, the identification of potential small molecule antiviral drugs is of particular importance. A critical antiviral target is the SARS-CoV-2 main protease (M pro ), and our aim was to identify lead compounds with potential inhibitory effects. We performed an initial molecular docking screen of 300 small molecules, which included phenolic compounds and fatty acids from our OliveNet™ library (224), and an additional group of curated pharmacological and dietary compounds. The prototypical α-ketoamide 13b inhibitor was used as a control to guide selection of the top 30 compounds with respect to binding affinity to the M pro active site. Further studies and analyses including blind docking were performed to identify hypericin, cyanidin-3-O-glucoside and SRT2104 as potential leads. Molecular dynamics simulations demonstrated that hypericin (ΔG = -18.6 and -19.3 kcal/mol), cyanidin-3-O-glucoside (ΔG = -50.8 and -42.1 kcal/mol), and SRT2104 (ΔG = -8.7 and -20.6 kcal/mol), formed stable interactions with the M pro active site. An enzyme-linked immunosorbent assay indicated that, albeit, not as potent as the covalent positive control (GC376), our leads inhibited the M pro with activity in the micromolar range, and an order of effectiveness of hypericin and cyanidin-3-O-glucoside > SRT2104 > SRT1720. Overall, our findings, and those highlighted by others indicate that hypericin and cyanidin-3-O-glucoside are suitable candidates for progress to in vitro and in vivo antiviral studies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The SARS-CoV-2 virus is causing COVID-19 resulting in an ongoing pandemic with serious health, social, and economic implications. Much research is focused in repurposing or identifying new small molecules which may interact with viral or host-cell molecular targets. An important SARS-CoV-2 target is the main protease (M pro ), and the peptidomimetic α-ketoamides represent prototypical experimental inhibitors. The protease is characterised by the dimerization of two monomers each which contains the catalytic dyad defined by Cys 145 and His 41 residues (active site). Dimerization yields the functional homodimer. Here, our aim was to investigate small molecules, including lopinavir and ritonavir, α-ketoamide 13b, and ebselen, for their ability to interact with the M pro . The sirtuin 1 agonist SRT1720 was also used in our analyses. Blind docking to each monomer individually indicated preferential binding of the ligands in the active site. Site-mapping of the dimeric protease indicated a highly reactive pocket in the dimerization region at the domain III apex. Blind docking consistently indicated a strong preference of ligand binding in domain III, away from the active site. Molecular dynamics simulations indicated that ligands docked both to the active site and in the dimerization region at the apex, formed relatively stable interactions. Overall, our findings do not obviate the superior potency with respect to inhibition of protease activity of covalently-linked inhibitors such as α-ketoamide 13b in the M pro active site. Nevertheless, along with those from others, our findings highlight the importance of further characterisation of the M pro active site and any potential allosteric sites., (Copyright © 2020 Elsevier Ltd. All rights reserved.)