Synthesis, in vitro and in silico evaluation of gallamide and selenogallamide derivatives as inhibitors of the SARS-CoV-2 main protease.

The present work reports the inhibitory effect of amides derived from gallic acid (gallamides) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro), along with cytotoxicity evaluation and molecular docking studies. In addition to gallamides, other relevant compounds were also synthesized and evaluated against Mpro, making a total of 25 compounds. Eight compounds presented solubility issues during the inhibitory assay and one showed no inhibitory activity. Compounds 3a, 3b, and 3f showed the highest enzymatic inhibition with IC50 = 0.26 ± 0.19 µM, 0.80 ± 0.38 µM, and 2.87 ± 1.17 µM, respectively. Selenogallamide 6a exhibited IC50 values of 5.42 ± 2.89 µM and a comparison with its nonselenylated congener 3c shows that the insertion of the chalcogen moiety improved the inhibitory capacity of the compound by approximately 10 times. Regarding the cellular toxicity in THP-1 and Vero cells, compounds 3e and 3g, showed moderate cytotoxicity in Vero cells, while for THP-1 both were nontoxic, with CC50 > 150 µM. Derivative 3d showed moderate cytotoxicity against both cell lines, whereas 6d was moderatly toxic to THP-1. Other compounds analyzed do not induce substantial cellular toxicity at the concentrations tested. The molecular docking results for compounds 3a, 3b, and 3f show that hydrogen bonding interactions involving the hydroxyl groups (OH) of the gallate moiety are relevant, as well as the carbonyl group.

Evaluation of in vitro antiviral activity of SARS-CoV-2 Mpro inhibitor pomotrelvir and cross-resistance to nirmatrelvir resistance substitutions.

The unprecedented scale of the COVID-19 pandemic and the rapid evolution of SARS-CoV-2 variants underscore the need for broadly active inhibitors with a high barrier to resistance. The coronavirus main protease (Mpro) is an essential cysteine protease required for viral polyprotein processing and is highly conserved across human coronaviruses. Pomotrelvir is a novel Mpro inhibitor that has recently completed a phase 2 clinical trial. In this report, we demonstrated that pomotrelvir is a potent competitive inhibitor of SARS-CoV-2 Mpro with high selectivity against human proteases. In the enzyme assay, pomotrelvir is also active against Mpro proteins derived from human coronaviruses CoV-229E, CoV-OC43, CoV-HKU1, CoV-NL63, MERS, and SARS-CoV. In cell-based SARS-CoV-2 replicon and SARS-CoV-2 infection assays, pomotrelvir has shown potent inhibitory activity and is broadly active against SARS-CoV-2 clinical isolates including Omicron variants. Many resistance substitutions of the Mpro inhibitor nirmatrelvir confer cross-resistance to pomotrelvir, consistent with the finding from our enzymatic analysis that pomotrelvir and nirmatrelvir compete for the same binding site. In a SARS-CoV-2 infection assay, pomotrelvir is additive when combined with remdesivir or molnupiravir, two nucleoside analogs targeting viral RNA synthesis. In conclusion, our results from the in vitro characterization of pomotrelvir antiviral activity support its further clinical development as an alternative COVID-19 therapeutic option.

Development of the Safe and Broad-Spectrum Aldehyde and Ketoamide Mpro inhibitors Derived from the Constrained α, γ-AA Peptide Scaffold.

SARS-CoV-2 is still wreaking havoc all over the world with surging morbidity and high mortality. The main protease (Mpro ) is essential in the replication of SARS-CoV-2, enabling itself an active target for antiviral development. Herein, we reported the design and synthesis of a new class of peptidomimetics-constrained α, γ-AA peptides, based on which a series of aldehyde and ketoamide inhibitors of the Mpro of SARS-CoV-2 were prepared. The lead compounds showed excellent inhibitory activity in the FRET-based Mpro enzymatic assay not only for the Mpro of SARS-CoV-2 but also for SARS-CoV and MERS-CoV, along with HCoVs like HCoV-OC43, HCoV-229E, HCoV-NL63 and HKU1. The X-ray crystallographic results demonstrated that our compounds form a covalent bond with the catalytic Cys145. They also demonstrated effective antiviral activity against live SARS-CoV-2. Overall, the results suggest that α, γ-AA peptide could be a promising molecular scaffold in designing novel Mpro inhibitors of SARS-CoV-2 and other coronaviruses.

Extending the Record of Terpenes from Soft Coral Sarcophyton mililatensis.

In the present study, a new polyoxygenated cembranoid named sarcomililatol H (1) as well as six known terpenes 2-7 with different skeletons were isolated from South China Sea soft coral Sarcophyton mililatensis. Based on the comprehensive analyses of 1D and 2D NMR spectroscopic data, the structure of the new compound 1 was established. This new cembranoid was characterized by the presence of the rarely encountered tetrahydropyran ring with the ether linkage across C-2 and C-12. By applying the time-dependent density functional theory electronic circular dichroism (TDDFT ECD) approach, the absolute configuration of sarcomililatol H (1) was determined. All of the isolates were subjected to the anti-inflammatory and anti-tumor bioassays. However, none of them was active in these evaluations. Additionally, the preliminary virtual screening of inhibitory against SARS-CoV-2 by molecular docking showed that diterpene 1 could be regarded as a SARS-CoV-2 main protease (Mpro ) inhibitor (binding energy: -7.63 kcal/mol). The discovery of these terpenes has expanded the chemical diversity and complexity of terpenes from the species S. mililatensis.

Structural Basis for the Inhibition of SARS-CoV-2 Mpro D48N Mutant by Shikonin and PF-07321332.

Preventing the spread of SARS-CoV-2 and its variants is crucial in the fight against COVID-19. Inhibition of the main protease (Mpro) of SARS-CoV-2 is the key to disrupting viral replication, making Mpro a promising target for therapy. PF-07321332 and shikonin have been identified as effective broad-spectrum inhibitors of SARS-CoV-2 Mpro. The crystal structures of SARS-CoV-2 Mpro bound to PF-07321332 and shikonin have been resolved in previous studies. However, the exact mechanism regarding how SARS-CoV-2 Mpro mutants impact their binding modes largely remains to be investigated. In this study, we expressed a SARS-CoV-2 Mpro mutant, carrying the D48N substitution, representing a class of mutations located near the active sites of Mpro. The crystal structures of Mpro D48N in complex with PF-07321332 and shikonin were solved. A detailed analysis of the interactions between Mpro D48N and two inhibitors provides key insights into the binding pattern and its structural determinants. Further, the binding patterns of the two inhibitors to Mpro D48N mutant and wild-type Mpro were compared in detail. This study illustrates the possible conformational changes when the Mpro D48N mutant is bound to inhibitors. Structural insights derived from this study will inform the development of new drugs against novel coronaviruses.

Substitutions in SARS-CoV-2 Mpro Selected by Protease Inhibitor Boceprevir Confer Resistance to Nirmatrelvir.

Nirmatrelvir, which targets the SARS-CoV-2 main protease (Mpro), is the first-in-line drug for prevention and treatment of severe COVID-19, and additional Mpro inhibitors are in development. However, the risk of resistance development threatens the future efficacy of such direct-acting antivirals. To gain knowledge on viral correlates of resistance to Mpro inhibitors, we selected resistant SARS-CoV-2 under treatment with the nirmatrelvir-related protease inhibitor boceprevir. SARS-CoV-2 selected during five escape experiments in VeroE6 cells showed cross-resistance to nirmatrelvir with up to 7.3-fold increased half-maximal effective concentration compared to original SARS-CoV-2, determined in concentration-response experiments. Sequence analysis revealed that escape viruses harbored Mpro substitutions L50F and A173V. For reverse genetic studies, these substitutions were introduced into a cell-culture-infectious SARS-CoV-2 clone. Infectivity titration and analysis of genetic stability of cell-culture-derived engineered SARS-CoV-2 mutants showed that L50F rescued the fitness cost conferred by A173V. In the concentration-response experiments, A173V was the main driver of resistance to boceprevir and nirmatrelvir. Structural analysis of Mpro suggested that A173V can cause resistance by making boceprevir and nirmatrelvir binding less favorable. This study contributes to a comprehensive overview of the resistance profile of the first-in-line COVID-19 treatment nirmatrelvir and can thus inform population monitoring and contribute to pandemic preparedness.

Molecular modeling study of tectoquinone and acteoside from Tectona grandis linn: a new SARS-CoV-2 main protease inhibitor against COVID-19.

Coronavirus disease 2019 (COVID-19), a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has drastically changed the lifestyle of people around the globe. Due to the lack of specific and effective antiviral drugs, transmission of the disease increases exponentially and makes it more serious and harder to control. Drugs that were assumed to be effective against COVID-19 have failed in various stages of clinical trials and this made the scientific community more disappointed. But, the race of researchers for developing new and effective antiviral to stop the disease progression still continues and our work is one among them. This study is an attempt to analyze the action of Tectoquinone and Acteoside; an important phytocompound, on SARS-CoV2 viral protease via in silico approach. The compounds were selected on the basis of their molecular docking values and they were subjected to molecular dynamics simulations about 50 ns to determine the stability and the thermodynamic feasibility between the target and the ligands. Binding energies like hydrogen bonding, hydrophobic and electrostatic interactions of the complexes were determined after MD simulations. The Pharmacokinetics and drug likeness evaluation of the compounds provide a strong evidence for the use of these compounds in developing drugs for clinical trials. Thus, the current study reveals the potential phytoconstituents present in Tectona grandis Linn to inhibit COVID-19 viral protease and thereby act as a lead therapeutic agent.Communicated by Ramaswamy H. Sarma.

Recent advances in small-molecular therapeutics for COVID-19.

The COVID-19 pandemic poses a fundamental challenge to global health. Since the outbreak of SARS-CoV-2, great efforts have been made to identify antiviral strategies and develop therapeutic drugs to combat the disease. There are different strategies for developing small molecular anti-SARS-CoV-2 drugs, including targeting coronavirus structural proteins (e.g. spike protein), non-structural proteins (nsp) (e.g. RdRp, Mpro, PLpro, helicase, nsp14, and nsp16), host proteases (e.g. TMPRSS2, cathepsin, and furin) and the pivotal proteins mediating endocytosis (e.g. PIKfyve), as well as developing endosome acidification agents and immune response modulators. Favipiravir and chloroquine are the anti-SARS-CoV-2 agents that were identified earlier in this epidemic and repurposed for COVID-19 clinical therapy based on these strategies. However, their efficacies are controversial. Currently, three small molecular anti-SARS-CoV-2 agents, remdesivir, molnupiravir, and Paxlovid (PF-07321332 plus ritonavir), have been granted emergency use authorization or approved for COVID-19 therapy in many countries due to their significant curative effects in phase III trials. Meanwhile, a large number of promising anti-SARS-CoV-2 drug candidates have entered clinical evaluation. The development of these drugs brings hope for us to finally conquer COVID-19. In this account, we conducted a comprehensive review of the recent advances in small molecule anti-SARS-CoV-2 agents according to the target classification. Here we present all the approved drugs and most of the important drug candidates for each target, and discuss the challenges and perspectives for the future research and development of anti-SARS-CoV-2 drugs.

Design and in silico investigation of novel Maraviroc analogues as dual inhibition of CCR-5/SARS-CoV-2 Mpro.

A sudden increase in life-threatening COVID-19 infections around the world inflicts global crisis and emotional trauma. In current study two druggable targets, namely SARS-COV-2 Mpro and CCR-5 were selected due to their significant nature in the viral life cycle and cytokine molecular storm respectively. The systematic drug repurposing strategy has been utilized to recognize inhibitory mechanism through extensive in silico investigation of novel Maraviroc analogues as promising inhibitors against SARS-CoV-2 Mpro and CCR-5. The dual inhibition specificity approach implemented in present study using molecular docking, molecular dynamics (MD), principal component analysis (PCA), free energy landscape (FEL) and MM/PBSA binding energy studies. The proposed Maraviroc analogues obtained from in silico investigation could be easily synthesized and constructive in developing significant drug against COVID-19 pandemic, with essentiality of their in vivo/in vitro evaluation to affirm the conclusions of this study. This will further fortify the concept of single drug targeting dual inhibition mechanism for treatment of COVID-19 infection and complications.

Review on development of potential inhibitors of SARS-CoV-2 main protease (MPro).

Background: The etiological agent for the coronavirus illness outbreak in 2019-2020 is a novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (COVID-19), whereas coronavirus disease pandemic of 2019 (COVID-19) has compelled the implementation of novel therapeutic options. Main body of the abstract: There are currently no targeted therapeutic medicines for this condition, and effective treatment options are quite restricted; however, new therapeutic candidates targeting the viral replication cycle are being investigated. The primary protease of the severe acute respiratory syndrome coronavirus 2 virus is a major target for therapeutic development (MPro). Severe acute respiratory syndrome coronavirus 2, severe acute respiratory syndrome coronavirus, and Middle East respiratory syndrome coronavirus (MERS-CoV) all seem to have a structurally conserved substrate-binding domain that can be used to develop novel protease inhibitors. Short conclusion: With the recent publication of the X-ray crystal structure of the severe acute respiratory syndrome coronavirus 2 Mm, virtual and in vitro screening investigations to find MPro inhibitors are fast progressing. The focus of this review is on recent advancements in the quest for small-molecule inhibitors of the severe acute respiratory syndrome coronavirus 2 main protease.

Appraisals of the Bangladeshi Medicinal Plant Calotropis gigantea Used by Folk Medicine Practitioners in the Management of COVID-19: A Biochemical and Computational Approach.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first recognized in Wuhan in late 2019 and, since then, had spread globally, eventually culminating in the ongoing pandemic. As there is a lack of targeted therapeutics, there is certain opportunity for the scientific community to develop new drugs or vaccines against COVID-19 and so many synthetic bioactive compounds are undergoing clinical trials. In most of the countries, due to the broad therapeutic spectrum and minimal side effects, medicinal plants have been used widely throughout history as traditional healing remedy. Because of the unavailability of synthetic bioactive antiviral drugs, hence all possible efforts have been focused on the search for new drugs and alternative medicines from different herbal formulations. In recent times, it has been assured that the Mpro, also called 3CLpro, is the SARS-CoV-2 main protease enzyme responsible for viral reproduction and thereby impeding the host's immune response. As such, Mpro represents a highly specified target for drugs capable of inhibitory action against coronavirus disease 2019 (COVID-19). As there continue to be no clear options for the treatment of COVID-19, the identification of potential candidates has become a necessity. The present investigation focuses on the in silico pharmacological activity of Calotropis gigantea, a large shrub, as a potential option for COVID-19 Mpro inhibition and includes an ADME/T profile analysis of that ligand. For this study, with the help of gas chromatography-mass spectrometry analysis of C. gigantea methanolic leaf extract, a total of 30 bioactive compounds were selected. Our analyses unveiled the top four options that might turn out to be prospective anti-SARS-CoV-2 lead molecules; these warrant further exploration as well as possible application in processes of drug development to combat COVID-19.

Structure-activity relationship (SAR) and molecular dynamics study of withaferin-A fragment derivatives as potential therapeutic lead against main protease (Mpro) of SARS-CoV-2.

The spread of novel coronavirus SARS-CoV-2 has directed to a state of an unprecedented global pandemic. Many synthetic compounds and FDA-approved drugs have been significantly inhibitory against the virus, but no SARS-CoV-2 solution has been identified. However, small molecule fragment-based derivatives of potent phytocompounds may serve as promising inhibitors against SARS-CoV-2. In the pursuit of exploring novel SARS-CoV-2 inhibitors, we generated small molecule fragment derivatives from potent phytocompounds using neural networking and machine learning-based tools, which can cover unexplored regions of the chemical space that still retain lead-like properties. Out of 300 derivative molecules from withaferin-A, hesperidin, and baicalin, 30 were screened out with synthetic accessibility scores > 4 having the best ADME properties. The withaferin-A derivative molecules 61 and 64 exhibited a significant binding affinity of - 7.84 kcal/mol and - 7.94 kcal/mol. The docking study reveals that withaferin-A mol 61 forms 5 polar H-bonds with the Mpro where amino acids involved are GLU166, THR190, CYS145, MET165, and GLN152 and upon QSAR analysis showed a minimal predicted IC50 value of 7762.47 nM. Furthermore, the in silico cytotoxicity predictions, pharmacophore modeling, and molecular dynamics simulation studies have resulted in predicting the highly potent small molecule derivative from withaferin-A (phytocompound from Withania somnifera) to be the potential inhibitor of SARS-CoV 2 protease (Mpro) and a promising future lead candidate against COVID-19. The rationale of choosing withaferin-A from Withania somnifera (Ashwagandha) was propelled by the innumerous applications of Ashwagandha for the treatment of various antiviral diseases, common cold, and fever since time immemorial. Graphical abstract.

In silico Analysis Revealed Potential Anti-SARS-CoV-2 Main Protease Activity by the Zonulin Inhibitor Larazotide Acetate.

The most severe outcome of COVID-19 infection is the development of interstitial pneumonia causing acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS), both responsible for the infected patients' mortality. ALI and ARDS are characterized by a leakage of plasma components into the lungs, compromising their ability to expand and optimally engage in gas exchange with blood, resulting in respiratory failure. We have previously reported that zonulin, a protein dictating epithelial and endothelial permeability in several districts, including the airways, is involved in ALI pathogenesis in mouse models, and that its peptide inhibitor Larazotide acetate (also called AT1001) ameliorated ALI and subsequent mortality by decreasing mucosal permeability to fluid and extravasation of neutrophils into the lungs. With the recent crystallographic resolution of the SARS-CoV-2 main protease (Mpro), an enzyme fundamental in the viral lifecycle, bound to peptidomimetic inhibitors N3 and 13b, we were able to perform molecular modeling investigation showing that AT1001 presents structural motifs similar to co-crystallized ligands. Specifically, molecular docking, MM-GBSA-based predictions and molecular dynamics showed that AT1001 docks extremely well in the Mpro catalytic domain through a global turn conformational arrangement without any unfavorable steric hindrance. Finally, we have observed that AT1001 can be superimposed onto the crystallized structures of N3 and 13b, establishing a higher number of interactions and accordingly a tighter binding. In vitro studies confirmed AT1001 anti-Mpro and preliminary investigation indicted an anti-viral activity. Combined, these studies suggest that AT1001, besides its well-demonstrated effect in ameliorating mucosal permeability in ALI/ARDS, may also exert a direct anti-SARS-CoV-2 effect by blocking the Mpro. AT1001 has been used extensively in a variety of animal models of ALI demonstrating robust safety and efficacy; it is currently in phase 3 trials in celiac subjects showing strong safety and efficacy profiles. We therefore propose its use as a specific anti-SARS-CoV-2 multitargeting treatment for the current pandemic.

Structure-Based Drug Design of an Inhibitor of the SARS-CoV-2 (COVID-19) Main Protease Using Free Software: A Tutorial for Students and Scientists.

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 (supplementary material) provides an illustrated step-by-step guide for the inhibitor design, 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.