In silico screening of potential compounds from begonia genus as 3CL protease (3Cl pro) SARS-CoV-2 inhibitors

Background: The emergence of Coronavirus disease (COVID-19) has been declared a pandemic and made a medical emergency worldwide. Various attempts have been made, including optimizing effective treatments against the disease or developing a vaccine. Since the SARS-CoV-2 protease crystal structure has been discovered, searching for its inhibitors by in silico technique becomes possible. Objective(s): This study aims to virtually screen the potential of phytoconstituents from the Begonia genus as 3Cl pro-SARS-CoV- 2 inhibitors, based on its crucial role in viral replication, hence making these proteases "promising" for the anti-SARS-CoV-2 target. Method(s): In silico screening was carried out by molecular docking on the web-based program DockThor and validated by a retrospective method. Predictive binding affinity (Dock Score) was used for scoring the compounds. Further molecular dynamics on Desmond was performed to assess the complex stability. Result(s): Virtual screening protocol was valid with the area under curve value 0.913. Molecular docking revealed only beta-sitosterol-3-O-beta-D-glucopyranoside with a lower docking score of -9.712 kcal/mol than positive control of indinavir. The molecular dynamic study showed that the compound was stable for the first 30 ns simulations time with Root Mean Square Deviation <3 A, despite minor fluctuations observed at the end of simulation times. Root Mean Square Fluctuation of catalytic sites HIS41 and CYS145 was 0.756 A and 0.773 A, respectively. Conclusion(s): This result suggests that beta-sitosterol-3-O-beta-Dglucopyranoside might be a prospective metabolite compound that can be developed as anti-SARS-CoV-2.Copyright © 2023, Page Press Publications. All rights reserved.

COMPOSITION OF LIPOPHILIC COMPONENTS OF RHODODENDRON ADAMSAY ETHER EXTRACT AND ACTIVITY AGAINST SARS-COV-2 MAIN PROTEASE

The composition of the lipophilic components of the Adams rhododendron RhododendronadamsiiRehd. Acid and neutral components were identified using gas-liquid chromatography with mass spectrometric detection. Methyl tert-butyl ether (MTBE), which has all the advantages of diethyl ether, but is devoid of its disadvantages, was used as the raw material extractant. It does not form peroxides and does not create increased gas contamination due to its higher boiling point. As a result, comparison with databases identified triterpene, phenolcarboxylic and aliphatic acids with a chain length of 8 to 30 carbon atoms, including saturated, unsaturated and dibasic acids. More than 150 triterpene and aliphatic components of the unsaponifiable residue and acid fractions have been identified. Previously, unsaponifiable residues of lipophilic extracts were practically not studied. Only components of Rh.adamsiiand other species of the genus rhododendron essential oils have been studied in detail. For bioactivity testing, samples of the whole extract and its fractionation products were prepared. Some of the studied samples show inhibitory activity against the main protease of SARS-CoV-2. © 2022 Altai State University. All rights reserved.

In silico Screening of the Potential Anti-SARS-CoV-2 Activities of Peptides from Vipera ammodytes ammodytes Venom by Molecular Docking

The coronavirus disease 2019 (COVID-19) is induced by the SARS-CoV-2 virus, which caused the global pandemic, infecting approximately 608.328.548 confirmed cases and bringing about 6.501.469 deaths worldwide, as WHO stated in September 2022. The disease is more deadly due to the lack of specific drug molecules or a treatment plan. Therefore, the development of potent pharmacological compounds is urgently required to combat COVID-19. Due to their biological actions, snake venoms constitute a source of potentially beneficial medicinal compounds. Vipera ammodytes ammodytes (VA) is a viper species whose venom has been shown to have anti-proliferative, antimetastatic, anti-cancer, and anti-microbial activities. This in silico study was conducted to evaluate the efficacy of selected VA venom proteins (Adamalysin II, Ammodytoxin A, Ammodytin L, L-amino acid oxidase) against molecular targets;Main protease (3CLpro) and Angiotensin-Converting Enzyme 2 (ACE2) by molecular docking study. Molecular docking investigations were performed by using AutoDock Vina software. All compounds displayed negative binding energy values to 3CLpro and ACE2, suggesting that their interactions with the active sites were favourable. L-amino acid oxidase had the highest binding affinity with both 3CLpro and ACE2. This study revealed for the first time that VA venom proteins are functional inhibitors of 3CLpro and ACE2 activities, and the components of VA venom can be considered potential SARS-CoV-2 inhibitors. However, more studies are needed to validate these compounds in vitro and in vivo. [ FROM AUTHOR] Copyright of Journal of the Institute of Science & Technology / Fen Bilimleri Estitüsü Dergisi is the property of Igdir University, Institute of Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Phytochemicals of Hibiscus sabdariffa with Therapeutic Potential against SARS-CoV-2: A Molecular Docking Study

In this study, the possible interactions of 17 phytochemicals that were reported as the most abundant biomolecules of Hibiscus sabdariffa, including many organic acids as well as catechin and quercetin derivatives, with 3CLpro and PLpro proteases of SARS-CoV-2 have been investigated via molecular docking. Caffeoylshikimic acid/3CLpro showed the lowest binding energy (-7.72 kcal/mol) with seven H-bonds. The second-lowest binding energy was computed in the chlorogenic acid/3CLpro complex (-7.18 kcal/mol), which was found to form 6 H-bonds. Also, low binding energies of cianidanol (-7.10 kcal/mol), cryptochlorogenic acid (-6.67 kcal/mol), and kaempferol (-6.82 kcal/mol) were calculated to 3CLpro with several H-bond interactions. Nelfinavir (-10.16 kcal/mol) and remdesivir (-6.40 kcal/mol), which have been used against COVID-19, were obtained to have low binding energies to 3CLpro with 3 H-bond formations each. On the other hand, the nicotiflorin/PLpro complex, which had the lowest binding energy (-7.40 kcal/mol), was found to have only 1 H-bond interaction. The secondlowest binding energy was reported in chlorogenic acid/PLpro (-7.20 kcal/mol), which was found to possess four H-bonds. On the other hand, epigallocatechin gallate/PLpro, which was shown to have a -5.95 kcal/mol binding energy, was found to form 8 H-bond interactions. Furthermore, the quercetin pentosylhexoside/PLpro complex was monitored to have low binding energy (-6.54 kcal/mol) with 9 H-bonds, which stands as the highest number of H-bonds in all complexes. Therefore, several molecules of Hibiscus sabdariffa were found to have strong binding affinity to the main proteases of SARS-CoV-2. This study suggests many compounds, including caffeoylshikimic acid and nicotiflorin, to inhibit 3CLpro and PLpro activities. As a result, numerous chemicals derived from Hibiscus sabdariffa have the potential to be employed therapeutically against SARS-CoV-2 infection. [ FROM AUTHOR] Copyright of Journal of the Institute of Science & Technology / Fen Bilimleri Estitüsü Dergisi is the property of Igdir University, Institute of Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Discovery of Chalcone-Based Hybrid Structures as High Affinity and Site-Specific Inhibitors against SARS-CoV-2: A Comprehensive Structural Analysis Based on Various Host-Based and Viral Targets.

Previous studies indicated that natural-based chalcones have significant inhibitory effects on the coronavirus enzymes 3CLpro and PLpro as well as modulation of some host-based antiviral targets (HBATs). In this study, a comprehensive computational and structural study was performed to investigate the affinity of our compound library consisting of 757 chalcone-based structures (CHA-1 to CHA-757) for inhibiting the 3CLpro and PLpro enzymes and against twelve selected host-based targets. Our results indicated that CHA-12 (VUF 4819) is the most potent and multi-target inhibitor in our chemical library over all viral and host-based targets. Correspondingly, CHA-384 and its congeners containing ureide moieties were found to be potent and selective 3CLpro inhibitors, and benzotriazole moiety in CHA-37 was found to be a main fragment for inhibiting the 3CLpro and PLpro. Surprisingly, our results indicate that the ureide and sulfonamide moieties are integral fragments for the optimum 3CLpro inhibition while occupying the S1 and S3 subsites, which is fully consistent with recent reports on the site-specific 3CLpro inhibitors. Finding the multi-target inhibitor CHA-12, previously reported as an LTD4 antagonist for the treatment of inflammatory pulmonary diseases, prompted us to suggest it as a concomitant agent for relieving respiratory symptoms and suppressing COVID-19 infection.

Identification of potential bioactive natural compounds from Indonesian medicinal plants against 3-chymotrypsin-like protease (3CLpro) of SARS-CoV-2: molecular docking, ADME/T, molecular dynamic simulations, and DFT analysis.

An outbreak of SARS-CoV-2 (COVID-19) has caused a global health emergency, resulting in hundreds of millions of infections and millions of deaths globally since December 2019. Due to the lack of a particular medicine or treatment approach and the fast spread of the virus around the world, it is imperative to find effective pharmacological molecules to combat the virus. Herein, we carried out docking-based virtual screening of selected 49 bioactive phytochemicals from 20 medicinal plants used in Jamu, an Indonesian traditional herbal medicine along with the 3CLpro inhibitor N3 towards the 3CLpro enzyme of SARS-CoV-2. From a total of 49 bioactive phytochemicals, eleven compounds exhibited good binding affinity against 3CLpro of SARS-CoV-2 (-7.2 to -8.5 kcal/mol). Accordingly, only seven phytochemicals fully obeyed drug-likeness properties. Ultimately, it was observed that both Luteolin and Naringenin have significant interactions with both of the catalytic residues of 3CLpro through hydrogen bonds and hydrophobic interactions, respectively. The drug-like characteristics of Luteolin and Naringenin were also confirmed by pharmacokinetic investigations. Further, an investigation into molecular dynamics (MD) simulations were undertaken to ensure the ligands would remain stable within the binding pocket. Finally, density-functional theory (DFT) calculations revealed the following order for biochemical reactivity: Naringenin > Luteolin > N3. The oxygen and hydrogen atom regions of these investigated ligands are suitable for electrophilic and nucleophilic attacks, respectively. These two bioactive phytochemicals from Tamarindus indica (Luteolin and Naringenin) as well as Citrus aurantifolia (Naringenin) might be potential antagonists of 3CLpro of SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Identification of Darunavir Derivatives for Inhibition of SARS-CoV-2 3CLpro.

The effective antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed around the world. The 3C-like protease (3CLpro) of SARS-CoV-2 plays a pivotal role in virus replication; it also has become an important therapeutic target for the infection of SARS-CoV-2. In this work, we have identified Darunavir derivatives that inhibit the 3CLpro through a high-throughput screening method based on a fluorescence resonance energy transfer (FRET) assay in vitro. We found that the compounds 29# and 50# containing polyphenol and caffeine derivatives as the P2 ligand, respectively, exhibited favorable anti-3CLpro potency with EC50 values of 6.3 µM and 3.5 µM and were shown to bind to SARS-CoV-2 3CLpro in vitro. Moreover, we analyzed the binding mode of the DRV in the 3CLpro through molecular docking. Importantly, 29# and 50# exhibited a similar activity against the protease in Omicron variants. The inhibitory effect of compounds 29# and 50# on the SARS-CoV-2 3CLpro warrants that they are worth being the template to design functionally improved inhibitors for the treatment of COVID-19.

No Substrate Left behind-Mining of Shotgun Proteomics Datasets Rescues Evidence of Proteolysis by SARS-CoV-2 3CLpro Main Protease.

Proteolytic processing is the most ubiquitous post-translational modification and regulator of protein function. To identify protease substrates, and hence the function of proteases, terminomics workflows have been developed to enrich and detect proteolytically generated protein termini from mass spectrometry data. The mining of shotgun proteomics datasets for such 'neo'-termini, to increase the understanding of proteolytic processing, is an underutilized opportunity. However, to date, this approach has been hindered by the lack of software with sufficient speed to make searching for the relatively low numbers of protease-generated semi-tryptic peptides present in non-enriched samples viable. We reanalyzed published shotgun proteomics datasets for evidence of proteolytic processing in COVID-19 using the recently upgraded MSFragger/FragPipe software, which searches data with a speed that is an order of magnitude greater than many equivalent tools. The number of protein termini identified was higher than expected and constituted around half the number of termini detected by two different N-terminomics methods. We identified neo-N- and C-termini generated during SARS-CoV-2 infection that were indicative of proteolysis and were mediated by both viral and host proteases-a number of which had been recently validated by in vitro assays. Thus, re-analyzing existing shotgun proteomics data is a valuable adjunct for terminomics research that can be readily tapped (for example, in the next pandemic where data would be scarce) to increase the understanding of protease function and virus-host interactions, or other diverse biological processes.

Key Allosteric and Active Site Residues of SARS-CoV-2 3CLpro Are Promising Drug Targets.

The main protease of SARS-CoV-2, 3-chymotrypsin-like protease (3CLpro), is a prominent target for antiviral development due to its essential role in the viral life cycle. Research has largely focused on competitive inhibitors of 3CLpro that target the active site. However, allosteric sites distal to the peptide substrate-binding region are also potential targets for the design of reversible noncompetitive inhibitors. Computational analyses have examined the importance of key contacts at allosteric sites of 3CLpro, but these contacts have not been validated experimentally. In this work, four druggable pockets spanning the surface of SARS-CoV-2 3CLpro were predicted: pocket 1 is the active site, whereas pockets 2, 3, and 4 are located away from the active site at the interface of domains II and III. Site-directed alanine mutagenesis of selected residues with important structural interactions revealed that 7 of 13 active site residues (N28, R40, Y54, S147, Y161, D187 and Q192) and 7 of 12 allosteric site residues (T111, R131, N133, D197, N203, D289 and D295) are essential for maintaining catalytically active and thermodynamically stable 3CLpro. Alanine substitution at these key amino acid residues inactivated or reduced the activity of 3CLpro. In addition, the thermodynamic stability of 3CLpro decreased in the presence of some of these mutations. This work provides experimental validation of essential contacts in the active and allosteric sites of 3CLpro that could be targeted with competitive and noncompetitive inhibitors as new therapeutics against COVID-19.

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

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

Geraniin as a potential inhibitor of SARS-CoV-2 3CLpro.

Geraniin is a polyphenolic compound first isolated from Geranium thunbergii. The major protease (Mpro), namely 3 C-like protease (3CLpro), of coronaviruses is considered an attractive drug target as it is essential for the processing and maturation of viral polyproteins. Thus, our primary goal is to explore the efficiency of geraniin on 3CLpro of SARS-CoV-2 using the computational biology strategy. In this work, we studied the anti-coronavirus effect of geraniin in vitro and its potential inhibitory mode against the 3CLpro of SARS-CoV-2. We found that geraniin inhibited HCoV-OC43 coronavirus-infected cells during the attachment and penetration phases. Molecular docking and dynamics simulations exhibited that geraniin had a strong binding affinity and high stable binding to 3CLpro of SARS-CoV-2. Geraniin showed a strong inhibitory activity on coronavirus and may be a potential inhibitor of SARS-CoV-2 3CLpro.

Mpro Inhibition: A comparative In silico Therapeutics from Native Indian Plant Species

One of the biggest healthcare threats of this century is COVID – 19, undoubtedly. It has caused millions of deaths and raised alerts in the healthcare domain. This study focuses on the importance of 10 native Indian plant species and the phytochemicals obtained from them as a potential inhibitor to the Main protease enzyme of SARS CoV-2. About 26 phytochemicals were shortlisted for the same from the selected plants. Molecular docking was used to analyze the binding affinity of the phytochemicals in the active pocket of the Main protease enzyme to assess their effectiveness. The docking scores resulted in the selection of four compounds being more favorable than the native inhibitor N3, namely Quercetin, Withaferin A, Sominone, and Nimbin, with their binding energies being-8.42,-9.21,-9.95,-8.88 kcal/mol respectively. Furthermore, these four were further analyzed for their bioavailability scores. The studies showed that Sominone, Withaferin A are more potent inhibitors to Mpro of the SARS CoV-2 in all four. Thus further in Vitro studies can be done accordingly for the same. © 2022 by the authors.

Harnessing and bioprospecting botanical-based herbal medicines against potential drug targets for COVID-19: a review coupled molecular docking studies.

Since the end of February 2020, the world has come to a standstill due to the virus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Since then, the global scientific community has explored various remedies and treatments against this virus, including natural products that have always been a choice because of their many benefits. Various known phytochemicals are well documented for their antiviral properties. Research is being carried out to discover new natural plant products or existing ones as a treatment measure for this disease. The three important targets in this regard are-papain like protease (PLpro), spike protein, and 3 chymotrypsin like proteases (3CLpro). Various docking studies are also being elucidated to identify the phytochemicals that modulate crucial proteins of the virus. The paper is simultaneously a comprehensive review that covers recent advances in the domain of the effect of various botanically derived natural products as an alternative treatment approach against Coronavirus Disease 2019 (COVID-19). Furthermore, the docking analyses revealed that rutin (inhibitor of the major protease of SARS-CoV-2), gallocatechin (e.g., interacting with 03 hydrogen bonds with a spike-like protein), lycorine (showing the best binding affinity with amino acids GLN498, THR500 and GLY446 of the spike-like protein), and quercetrin (inhabiting at its residues ASP216, PHE219, and ILE259) are promising inhibitors of SARS­CoV­2.Communicated by Ramaswamy H. Sarma.

Discovery of 3CLpro inhibitor of SARS-CoV-2 main protease.

Coronavirus main protease (3CLpro), a special cysteine protease in coronavirus family, is highly desirable in the life cycle of coronavirus. Here, molecular docking, ADMET pharmacokinetic profiles and molecular dynamics (MD) simulation were performed to develop specific 3CLpro inhibitor. The results showed that the 137 compounds originated from Chinese herbal have good binding affinity to 3CLpro. Among these, Cleomiscosin C, (+)-Norchelidonine, Protopine, Turkiyenine, Isochelidonine and Mallotucin A possessed prominent drug-likeness properties. Cleomiscosin C and Turkiyenine exhibited excellent pharmacokinetic profiles. Furthermore, the complex of Cleomiscosin C with SARS-CoV-2 main protease presented high stability. The findings in this work indicated that Cleomiscosin C is highly promising as a potential 3CLpro inhibitor, thus facilitating the development of effective drugs for COVID-19.

In this work, computer aided drug design technology was used to study the main protease 3CLpro of novel coronavirus, and functional small molecules with inhibitory effects on novel coronavirus were screened from the compound library of natural products. The results showed that Cleomiscosin C is highly promising as a potential 3CLpro inhibitor with prominent binding affinity, pharmacokinetic profiles and stability.

Exploring potential SARS-CoV-2 Mpro non-covalent inhibitors through docking, pharmacophore profile matching, molecular dynamic simulation, and MM-GBSA.

CONTEXT: In the replication of SARS-CoV-2, the main protease (Mpro/3CLpro) is significant. It is conserved in a number of novel coronavirus variations, and no known human proteases share its cleavage sites. Therefore, 3CLpro is an ideal target. In the report, we screened five potential inhibitors (1543, 2308, 3717, 5606, and 9000) of SARS-CoV-2 Mpro through a workflow. The calculation of MM-GBSA binding free energy showed that three of the five potential inhibitors (1543, 2308, 5606) had similar inhibitor effects to X77 against Mpro of SARS-CoV-2. In conclusion, the manuscript lays the groundwork for the design of Mpro inhibitors. METHODS: In the virtual screening phase, we used structure-based virtual screening (Qvina2.1) and ligand-based virtual screening (AncPhore). In the molecular dynamic simulation part, we used the Amber14SB + GAFF force field to perform molecular dynamic simulation of the complex for 100 ns (Gromacs2021.5) and performed MM-GBSA binding free energy calculation according to the simulation trajectory.

Molecular Networking Accelerated Discovery of Biflavonoid Alkaloids from Cephalotaxus sinensis.

Four undescribed biflavonoid alkaloids, sinenbiflavones A-D, were isolated from Cephalotaxus sinensis using a MS/MS-based molecular networking guided strategy. Their structures were elucidated by series of spectroscopic methods (HR-ESI-MS, UV, IR, 1D, and 2D NMR). Sinenbiflavones A-D are the first examples of amentoflavone-type (C-3'-C-8'') biflavonoid alkaloids. Meanwhile, sinenbiflavones B and D are the unique C-6-methylated amentoflavone-type biflavonoid alkaloids. Sinenbiflavone D showed weak SARS-CoV-2 3CLpro inhibitory activity with 43 % inhibition rate at 40 µM.

Ferrocenoyl-substituted quinolinone and coumarin as organometallic inhibitors of SARS-CoV-2 3CLpro main protease.

The 3-chymotrypsin-like protease 3CLpro from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a potential target for antiviral drug development. In this work, three organometallic ferrocene-modified quinolinones and coumarins were compared to their benzoic acid ester analogues with regard to inhibition of 3CLpro using an HPLC-based assay with a 15mer model peptide as the substrate. In contrast to FRET-based assays, this allows direct identification of interference of buffer constituents with the inhibitors, as demonstrated by the complete abolishment of ebselen inhibitory activity in the presence of dithiothreitol as a redox protectant. The presence of the organometallic ferrocene moiety significantly increased the stability of the title compounds towards hydrolysis. Among the studied compounds, 4-ferrocenyloxy-1-methyl-quinol-2-one was identified as the most stable and potent inhibitor candidate. IC50 values determined for ebselen and this sandwich complex compound are (0.40 ± 0.07) and (2.32 ± 0.21) µM, respectively.

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

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

Discovery and characterization of the covalent SARS-CoV-2 3CLpro inhibitors from Ginkgo biloba extract via integrating chemoproteomic and biochemical approaches.

BACKGROUND: The 3C-like proteases (3CLpros) are cysteine-rich homodimeric proteins and can be covalently modified by numerous natural and synthetic compounds, which in turn, block the proteolytic activity or the formation of enzymatically active dimeric forms. Although herbal medicines have been widely used to treat COVID-19, identification of the key herbal constituents that can covalently modify the 3CLpros in ß-coronaviruses (CoVs) remains a big challenge. AIMS: To construct a comprehensive approach for efficient discovering the covalent SARS-CoV-2 3CLpro inhibitors from herbal medicines. To decipher the key anti-SARS-CoV-2 3CLpro constituents in Ginkgo biloba extract 50 (GBE50) and to study their anti-SARS-CoV-2 3CLpro mechanisms. METHODS: SARS-CoV-2 3CLpro inhibition assay including time-dependent inhibition assays and inactivation kinetic analyses were conducted using a fluorescence-based biochemical assay. The constituents in GBE50 were analyzed by UHPLC-Q-Exactive Orbitrap HRMS. The peptides modified by herbal constituents were characterized by using nanoLC-MS/MS. RESULTS: Following testing the anti-SARS-CoV-2 3CLpro effects of 104 herbal medicines, it was found that Ginkgo biloba extract 50 (GBE50) potently inhibited SARS-CoV-2 3CLpro in dose- and time-dependent manners. A total of 38 constituents were identified from GBE50 by UHPLC-Q-Exactive Orbitrap HRMS, while 26 peptides modified by 18 constituents were identified by chemoproteomic profiling. The anti-SARS-CoV-2 3CLpro effects of 18 identified covalent inhibitors were then validated by performing time-dependent inhibition assays. The results clearly demonstrated that most tested constituents showed time-dependent inhibition on SARS-CoV-2 3CLpro, while gallocatechin and sciadopitysin displayed the most potent anti-SARS-CoV-2 3CLpro effects. CONCLUSION: Collectively, GBE50 and some constituents in this herbal product could strongly inhibit SARS-CoV-2 3CLpro in dose- and time-dependent manner. Gallocatechin and sciadopitysin were identified as potent SARS-CoV-2 3CLpro inhibitors, which offers promising lead compounds for the development of novel anti-SARS-CoV-2 drugs.

Would the Development of a Multitarget Inhibitor of 3CLpro and TMPRSS2 be Promising in the Fight Against SARS-CoV-2?

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), responsible for generating COVID-19, has spread worldwide and was declared a pandemic by the World Health Organization (WHO) on 11 March 2020, being responsible for various damages to public health, social life, and the economy of countries. Its high infectivity and mutation rates have stimulated researchers and pharmaceutical companies to search for new therapies against this disease. These efforts resulted in several vaccines and the identification of Molnupiravir as an oral treatment against this disease. However, identifying new alternatives and critical information is necessary to fight against this devastating agent. The findings in recent years regarding the structure and biochemistry of SARS-CoV2 are remarkable. In anti-CoV drug discovery, various targets such as structural, non-structural, and host-related proteins are explored. In fact, 3CLpro is the most used among non-structural proteins since this protease cleaves peptide sequences after the glutamine residue, and no human protease has this function. This makes this macromolecule an excellent drug target for discovering new compounds. Another promising target is the transmembrane protease serine 2 (TMPRSS2). Recent studies point to TMPRSS2 as one of the main targets responsible for viral entry, related to the cleavage of the S protein. Similar to cathepsins, TMPRSS2 is also responsible for cleaving the spike protein SARS-CoV2, which binds to the ACE2 receptor. Thus, TMPRSS2 is one of the targets that may represent new alternatives in treating SARS-CoV2. In this context, would discovering a multitarget inhibitor be the new strategy in searching for drugs against SARS-CoV2? For many years, new drug discovery was based on the "one drug, one target" premise, where the biological action is related to interactions with only one biological target. However, this paradigm has been overcome as new evidence of multiple mechanisms of action for a single drug. Finally, this review will present a perspective on drug design based on a multitarget strategy against 3CLpro and TMPRSS2. We hope to provide new horizons for researchers worldwide searching for more effective drugs against this devastating agent.