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.

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.

Virtual screening campaigns and ADMET evaluation to unlock the potency of flavonoids from Erythrina as 3CLpro SARS-COV-2 inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic with more than 6 million deaths worldwide. Flavonoids from the genus Erythrina may inhibit SARS-CoV-2, targeting 3C-like protease (3CLpro), an enzyme essential in the virus's growth. Hence, this study aimed to screen 378 flavonoids from Erythrina against 3CLpro, using molecular docking, Lipinski's rule of five, and in silico absorption, distribution, metabolism, excretion, and toxicity. These virtual screening campaigns suggest that 108 flavonoids have stronger binding energy values (−13.23 to −10.20 kcal/mol) than the N3 inhibitor (−10.14 kcal/mol) as the reference ligand. Some 33 of these flavonoids may be hepatoxicity and mutagenicity-free. They are also non-hERG I and II inhibitors. Two of them, orientanol E (171) and erycaffra F (57), have binding energy values in the top 10 hits and good absorption profiles, despite their poor distribution properties. They may have a high bioavailability in the body and be excreted from the body through feces. Conducted molecular dynamics simulations also support orientanol E (171) and erycaffra F (57) as 3CLpro inhibitor candidates. Our study suggests that flavonoids from Erythrina have potential as 3CLpro inhibitors, which help guide further in vitro and in vivo experiments in COVID-19 drug development © 2023 Vicki Nishinarizki et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/)

Virtual Screening of the Flavonoids Compounds with the SARS-CoV-2 3C-like Protease as the Lead Compounds for the COVID-19

As of 1st of September 2020, the COVID-19 pandemic has reached an unprecedented level of more than 25 million cases with more than 850,000 deaths. Moreover, all the drug candidates are still undergoing testing in clinical trials. In this regard, a breakthrough in drug design is neces-sary. One strategy to devise lead compounds is leveraging natural products as a lead source. Sever-al companies and research institutes are currently developing anti-SARS-CoV-2 lead from natural products. Flavonoids are well known as a class of antiviral compounds library. The objective of this research is to employ virtual screening methods for obtaining the best lead compounds from the library of flavonoid compounds. This research employed virtual screening methods that com-prised of downloading the protein and lead compound structures, QSAR analysis prediction, itera-tions of molecular docking simulation, and ADME-TOX simulation for toxicity prediction. The QSAR analysis found that the tested compounds have broad-spectrum antiviral activity, and some of them exhibit specific binding to the 3C-like Protease of the Coronavirus. Moreover, juglanin was found as the compound with the fittest binding with the Protease enzyme of SARS-CoV-2. Al-though most of the tested compounds are deemed toxic by the ADME-Tox test, further research should be conducted to comprehend the most feasible strategy to deliver the drug to the infected lung cells. The juglanin compound is selected as the fittest candidate as the SARS-CoV-2 lead compound in the tested flavonoid samples. However, further research should be conducted to observe the lead delivery method to the cell.Copyright © 2021 Bentham Science Publishers.

Elucidation of the inhibitory activity of plant-derived SARS-CoV inhibitors and their potential as SARS-CoV-2 inhibitors.

Several drugs are now being tested as possible therapies due to the necessity of treating SARS-CoV-2 infection. Although approved vaccines bring much hope, a vaccination program covering the entire global population will take a very long time, making the development of effective antiviral drugs an effective solution for the immediate treatment of this dangerous infection. Previous studies found that three natural compounds, namely, tannic acid, 3-isotheaflavin-3-gallate and theaflavin-3,3-digallate, are effective proteinase (3CLpro) inhibitors of SARS-CoV (IC50 <10 µM). Based on this information and due to the high sequence identity between SARS-CoV and SARS-CoV-2 3CLpro, these three compounds could be candidate inhibitors of SARS-CoV-2 3CLpro. This paper explores the structural and energetic features that guided the molecular recognition of these three compounds for dimeric SARS-CoV-2 and SARS-CoV 3CLpro, the functional state of this enzyme, using docking and MD simulations with the molecular mechanics-generalized-born surface area (MMGBSA) approach. Energetic analysis demonstrated that the three compounds reached good affinities in both systems in the following order: tannic acid > 3-isotheaflavin-3-gallate > theaflavin-3,3-digallate. This tendency is in line with that experimentally reported between these ligands and SARS-CoV 3CLpro. Therefore, tannic acid may have clinical usefulness against COVID-19 by acting as a potent inhibitor of SARS-CoV-2 3CLpro.Communicated by Ramaswamy H. Sarma.

Multidimensional virtual screening approaches combined with drug repurposing to identify potential covalent inhibitors of SARS-CoV-2 3CL protease.

The outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused an unprecedented global pandemic, and new cases are still on the rise due to the absence of effective medicines. However, developing new drugs within a short time is extremely difficult. Repurposing the existing drugs provides a fast and effective strategy to identify promising inhibitors. Here we focus on the screening of drugs database for discovering potential covalent inhibitors that target 3-chymotrypsin-like protease (3CLpro), an essential enzyme mediating viral replication and transcription. Firstly, we constructed a receptor-ligand pharmacophore model and verified it through decoy set. The importance of pharmacophore features was evaluated by combining molecular dynamics simulation with interaction analyses. Then, covalent docking was used to perform further screening. According to docking score and Prime/Molecular Mechanics Generalized Born Surface Area (MM-GBSA) score, total ten compounds obtained good scores and successfully established covalent bonds with the catalytic Cys145 residue. They also formed favorable interactions with key residues in active sites and closely integrated with 3CLpro with binding modes similar to known 3CLpro inhibitor. Finally, the top four hits DB08732, DB04653, DB01871 and DB07299 were further subjected to 100 ns molecular dynamics (MD) simulation and MM-GBSA binding free energy calculations. The results suggest that the four candidates show good binding affinities for 3CLpro, which warrants further evaluation for their in-vitro/in-vivo activities. Overall, our research methods provide a valuable reference for discovering promising inhibitors against SARS-CoV-2 and help to fight against the epidemic.Communicated by Ramaswamy H. Sarma.

Synthesis of conjugates of (aR,7S)-colchicine with monoterpenoids and investigation of their biological activity.

Conjugates of the natural alkaloid (aR,7S)-colchicine with bicyclic monoterpenoids and their derivatives were synthesized for the first time. Molecular docking of the synthesized agents in the active site of the main viral protease of the SARS-CoV-2 virus was carried out. The cytotoxic properties of the agents against different cell lines and the ability to inhibit the main viral protease 3CLPro were studied.

Flavonoids from the roots and rhizomes of Sophoratonkinensis and their in vitro anti-SARS-CoV-2 activity.

Acute respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had caused a global pandemic since 2019, and posed a serious threat to global health security. Traditional Chinese medicine (TCM) has played an indispensable role in the battle against the epidemic. Many components originated from TCMs were found to inhibit the production of SARS-CoV-2 3C-like protease (3CLpro) and papain-like protease (PLpro), which are two promising therapeutic targets to inhibit SARS-CoV-2. This study describes a systematic investigation of the roots and rhizomes of Sophora tonkinensis, which results in the characterization of 12 new flavonoids, including seven prenylated flavanones (1-7), one prenylated flavonol (8), two prenylated chalcones (9-10), one isoflavanone (11), and one isoflavan dimer (12), together with 43 known compounds (13-55). Their structures including the absolute configurations were elucidated by comprehensive analysis of MS, 1D and 2D NMR data, and time-dependent density functional theory electronic circular dichroism (TDDFT ECD) calculations. Compounds 12 and 51 exhibited inhibitory effects against SARS-CoV-2 3CLpro with IC50 values of 34.89 and 19.88 µmol·L-1, repectively while compounds 9, 43 and 47 exhibited inhibitory effects against PLpro with IC50 values of 32.67, 79.38, and 16.74 µmol·L-1, respectively.

Chetomin, a SARS-CoV-2 3C-like Protease (3CLpro) Inhibitor: In Silico Screening, Enzyme Docking, Molecular Dynamics and Pharmacokinetics Analysis.

The emergence of the Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to over 6 million deaths. The 3C-like protease (3CLpro) enzyme of the SARS-CoV-2 virus is an attractive druggable target for exploring therapeutic drug candidates to combat COVID-19 due to its key function in viral replication. Marine natural products (MNPs) have attracted considerable attention as alternative sources of antiviral drug candidates. In looking for potential 3CLpro inhibitors, the MNP database (>14,000 molecules) was virtually screened against 3CLpro with the assistance of molecular docking computations. The performance of AutoDock and OEDocking software in anticipating the ligand-3CLpro binding mode was first validated according to the available experimental data. Based on the docking scores, the most potent MNPs were further subjected to molecular dynamics (MD) simulations, and the binding affinities of those molecules were computed using the MM-GBSA approach. According to MM-GBSA//200 ns MD simulations, chetomin (UMHMNP1403367) exhibited a higher binding affinity against 3CLpro than XF7, with ΔGbinding values of −55.5 and −43.7 kcal/mol, respectively. The steadiness and tightness of chetomin with 3CLpro were evaluated, revealing the high stabilization of chetomin (UMHMNP1403367) inside the binding pocket of 3CLpro throughout 200 ns MD simulations. The physicochemical and pharmacokinetic features of chetomin were also predicted, and the oral bioavailability of chetomin was demonstrated. Furthermore, the potentiality of chetomin analogues −namely, chetomin A-D− as 3CLpro inhibitors was investigated. These results warrant further in vivo and in vitro assays of chetomin (UMHMNP1403367) as a promising anti-COVID-19 drug candidate.

Insights into the Dynamics and Binding of Two Polyprotein Substrate Cleavage Points in the Context of the SARS-CoV-2 Main and Papain-like Proteases.

It is well known that vital enzymes in the replication process of the coronavirus are the SARS-CoV-2 PLpro and SARS-CoV-2 3CLpro, both of which are important targets in the search for anti-coronavirus agents. These two enzymes are responsible for cleavage at various polyprotein sites in the SARS-CoV-2 lifecycle. Herein, the dynamics of the polyprotein cleavage sequences for the boundary between non-structural proteins Nsp1 and Nsp2 (CS1) and between Nsp2 and Nsp3 (CS2) in complex with both the papain-like protein PLpro and the main protease 3CLpro were explored using computational methods. The post dynamics analysis reveals that CS1 and CS2 both have greater stability when complexed with PLpro. Of these two, greater stability is observed for the CS1-PLpro complex, while destabilization resulting in loss of CS2 from the PLpro active site is observed for CS2-PLpro, suggesting the rate of exchange by the papain-like protease is faster for CS2 compared to CS1. On the other hand, the 3CLpro main protease also reveals stability for CS1 suggesting that the main protease could also play a potential role in the cleavage at point CS1. However, destabilization occurs early in the simulation for the complex CLpro-CS2 suggesting a poor interaction and non-plausible protease cleavage of the polyprotein at CS2 by the main protease. These findings could be used as a guide in the development and design of potent COVID-19 antiviral inhibitors that mimic the CS1 cleavage site.

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

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

SARS-CoV-2 main protease (3CLpro) interaction with acyclovir antiviral drug/methyl-ß-cyclodextrin complex: Physiochemical characterization and molecular docking.

During the current outbreak of the novel coronavirus disease 2019 (COVID-19), researchers have examined several antiviral drugs with the potential to inhibit the proliferation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The antiviral drug acyclovir (AVR), which is used to treat COVID-19, in complex with methyl-ß-cyclodextrin (Mß-CD) was examined in the solution and solid phases. UV-visible and fluorescence spectroscopic analyses confirmed that the guest (AVR) was included inside the host (Mß-CD) cavity. A solid inclusion complex of AVR was prepared by co-precipitation, physical mixing, kneading, and bath sonication methods at a 1:1 ratio of Mß-CD:AVR. The prepared Mß-CD:AVR inclusion complex was characterized using Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM) analysis. Phase solubility studies indicated the Mß-CD:AVR inclusion complex exhibited a higher stability constant and linear enhancement in AVR solubility with increasing Mß-CD concentrations. In silico analysis of the Mß-CD/AVR inclusion complex confirmed that AVR drugs show potential as inhibitors of SARS-CoV-2 3C-like protease (3CLpro) receptors. Results obtained using the PatchDock and FireDock servers indicated that the most favorable docking ligand was Mß-CD:AVR, which interacted with SARS-CoV-2 (3CLPro) protease inhibitors with high geometric shape complementarity scores (2522 and 5872) and atomic contact energy (-313.77 and -214.70 kcal mol-1). Our results suggest that the Mß-CD/AVR inclusion complex inhibits the main protease of SARS-CoV-2, although further wet-lab experiments are needed to verify these findings.

Rational identification of small molecules derived from 9,10-dihydrophenanthrene as potential inhibitors of 3CLpro enzyme for COVID-19 therapy: a computer-aided drug design approach.

Small molecules such as 9,10-dihydrophenanthrene derivatives have remarkable activity toward inhibition of SARS-CoV-2 3CLpro and COVID-19 proliferation, which show a strong correlation between their structures and bioactivity. Therefore, these small compounds could be suitable for clinical pharmaceutical use against COVID-19. The objective of this study was to remodel the structures of 9,10-dihydrophenanthrene derivatives to achieve a powerful biological activity against 3CLpro and favorable pharmacokinetic properties for drug design and discovery. Therefore, by the use of bioinformatics techniques, we developed robust 3D-QSAR models that are capable of describing the structure-activity relationship for 46 molecules based on 9,10-dihydrophenanthrene derivatives using CoMFA/SE (R 2 = 0.97, Q 2 = 0.81, R 2 pred = 0.95, c R 2 p = 0.71) and CoMSIA/SEHDA (R 2 = 0.94, Q 2 = 0.76, R 2 pred = 0.91, c R 2 p = 0.65) techniques. Accordingly, 96 lead compounds were generated based on a template molecule that showed the highest observed activity in vitro (T40, pIC50 = 5.81) and predicted their activities and bioavailability in silico. The rational screening outputs of 3D-QSAR, Molecular docking, ADMET, and MM-GBSA led to the identification of 9 novel modeled molecules as potent noncovalent drugs against SARS-CoV-2-3CLpro. Finally, by molecular dynamics simulations, the stability and structural dynamics of 3CLpro free and complex (PDB code: 6LU7) were discussed in the presence of samples of 9,10-dihydrophenanthrene derivative in an aqueous environment. Overall, the retrosynthesis of the proposed drug compounds in this study and the evaluation of their bioactivity in vitro and in vivo may be interesting for designing and discovering a new drug effective against COVID-19. Supplementary Information: The online version contains supplementary material available at 10.1007/s11224-022-02004-z.

Discovery of 9,10-dihydrophenanthrene derivatives as SARS-CoV-2 3CLpro inhibitors for treating COVID-19.

The epidemic coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now spread worldwide and efficacious therapeutics are urgently needed. 3-Chymotrypsin-like cysteine protease (3CLpro) is an indispensable protein in viral replication and represents an attractive drug target for fighting COVID-19. Herein, we report the discovery of 9,10-dihydrophenanthrene derivatives as non-peptidomimetic and non-covalent inhibitors of the SARS-CoV-2 3CLpro. The structure-activity relationships of 9,10-dihydrophenanthrenes as SARS-CoV-2 3CLpro inhibitors have carefully been investigated and discussed in this study. Among all tested 9,10-dihydrophenanthrene derivatives, C1 and C2 display the most potent SARS-CoV-2 3CLpro inhibition activity, with IC50 values of 1.55 ± 0.21 µM and 1.81 ± 0.17 µM, respectively. Further enzyme kinetics assays show that these two compounds dose-dependently inhibit SARS-CoV-2 3CLprovia a mixed-inhibition manner. Molecular docking simulations reveal the binding modes of C1 in the dimer interface and substrate-binding pocket of the target. In addition, C1 shows outstanding metabolic stability in the gastrointestinal tract, human plasma, and human liver microsome, suggesting that this agent has the potential to be developed as an orally administrated SARS-CoV-2 3CLpro inhibitor.

Ligand-based quantitative structural assessments of SARS-CoV-2 3CLpro inhibitors: An analysis in light of structure-based multi-molecular modeling evidences.

Due to COVID-19, the whole world is undergoing a devastating situation, but treatment with no such drug candidates still has been established exclusively. In that context, 69 diverse chemicals with potential SARS-CoV-2 3CLpro inhibitory property were taken into consideration for building different internally and externally validated linear (SW-MLR and GA-MLR), non-linear (ANN and SVM) QSAR, and HQSAR models to identify important structural and physicochemical characters required for SARS-CoV-2 3CLpro inhibition. Importantly, 2-oxopyrrolidinyl methyl and benzylester functions, and methylene (hydroxy) sulphonic acid warhead group, were crucial for retaining higher SARS-CoV-2 3CLpro inhibition. These GA-MLR and HQSAR models were also applied to predict some already repurposed drugs. As per the GA-MLR model, curcumin, ribavirin, saquinavir, sepimostat, and remdesivir were found to be the potent ones, whereas according to the HQSAR model, lurasidone, saquinavir, lopinavir, elbasvir, and paritaprevir were the highly effective SARS-CoV-2 3CLpro inhibitors. The binding modes of those repurposed drugs were also justified by the molecular docking, molecular dynamics (MD) simulation, and binding energy calculations conducted by several groups of researchers. This current work, therefore, may be able to find out important structural parameters to accelerate the COVID-19 drug discovery processes in the future.

Flavonoids in Ampelopsis grossedentata as covalent inhibitors of SARS-CoV-2 3CLpro: Inhibition potentials, covalent binding sites and inhibitory mechanisms.

Coronavirus 3C-like protease (3CLpro) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CLpro inhibitory activity, but the key constituents with SARS-CoV-2-3CLpro inhibitory effect and their mechanisms were unrevealed. Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide profiling and time-dependent biochemical assay, was applied to identify the crucial constituents in AGE and to uncover their inhibitory mechanisms. The results demonstrated that the flavonoid-rich fractions (10-17.5 min) displayed strong SARS-CoV-2-3CLpro inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CLpro inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CLpro in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CLpro at Cys300 and Cys44, while dihydromyricetin and isodihydromyricetin covalently bound at Cys300. Covalent docking coupling with molecular dynamics simulations showed the detailed interactions between the orthoquinone form of myricetin and two covalent binding sites (surrounding Cys300 and Cys44) of SARS-CoV-2 3CLpro. Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CLpro, while the newly identified SARS-CoV-2 3CLpro inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.

Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CLpro substrate degradome.

The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. We delineate the human protein substrate landscape of 3CLpro 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 3CLpro 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 3CLpro 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 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design.

Identification of Vitamin K3 and its analogues as covalent inhibitors of SARS-CoV-2 3CLpro.

After the emergence of the pandemic, repurposed drugs have been considered as a quicker way of finding potential antiviral agents. SARS-CoV-2 3CLpro is essential for processing the viral polyproteins into mature non-structural proteins, making it an attractive target for developing antiviral agents. Here we show that Vitamin K3 screened from the FDA-Approved Drug Library containing an array of 1,018 compounds has potent inhibitory activity against SARS-CoV-2 3CLpro with the IC50 value of 4.78 ± 1.03 µM, rather than Vitamin K1, K2 and K4. Next, the time-dependent inhibitory experiment was carried out to confirm that Vitamin K3 could form the covalent bond with SARS-CoV-2 3CLpro. Then we analyzed the structure-activity relationship of Vitamin K3 analogues and identified 5,8-dihydroxy-1,4-naphthoquinone with 9.8 times higher inhibitory activity than Vitamin K3. Further mass spectrometric analysis and molecular docking study verified the covalent binding between Vitamin K3 or 5,8-dihydroxy-1,4-naphthoquinone and SARS-CoV-2 3CLpro. Thus, our findings provide valuable information for further optimization and design of novel inhibitors based on Vitamin K3 and its analogues, which may have the potential to fight against SARS-CoV-2.

Potential In Vitro Inhibition of Selected Plant Extracts against SARS-CoV-2 Chymotripsin-Like Protease (3CLPro) Activity.

Antiviral treatments inhibiting Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication may represent a strategy complementary to vaccination to fight the ongoing Coronavirus disease 19 (COVID-19) pandemic. Molecules or extracts inhibiting the SARS-CoV-2 chymotripsin-like protease (3CLPro) could contribute to reducing or suppressing SARS-CoV-2 replication. Using a targeted approach, we identified 17 plant products that are included in current and traditional cuisines as promising inhibitors of SARS-CoV-2 3CLPro activity. Methanolic extracts were evaluated in vitro for inhibition of SARS-CoV-2 3CLPro activity using a quenched fluorescence resonance energy transfer (FRET) assay. Extracts from turmeric (Curcuma longa) rhizomes, mustard (Brassica nigra) seeds, and wall rocket (Diplotaxis erucoides subsp. erucoides) at 500 µg mL-1 displayed significant inhibition of the 3CLPro activity, resulting in residual protease activities of 0.0%, 9.4%, and 14.9%, respectively. Using different extract concentrations, an IC50 value of 15.74 µg mL-1 was calculated for turmeric extract. Commercial curcumin inhibited the 3CLPro activity, but did not fully account for the inhibitory effect of turmeric rhizomes extracts, suggesting that other components of the turmeric extract must also play a main role in inhibiting the 3CLPro activity. Sinigrin, a major glucosinolate present in mustard seeds and wall rocket, did not have relevant 3CLPro inhibitory activity; however, its hydrolysis product allyl isothiocyanate had an IC50 value of 41.43 µg mL-1. The current study identifies plant extracts and molecules that can be of interest in the search for treatments against COVID-19, acting as a basis for future chemical, in vivo, and clinical trials.

Identification of Novel Drug Candidates for the Inhibition of Catalytic Cleavage Activity of Coronavirus 3CL-like Protease Enzyme.

BACKGROUND: There has been tremendous pressure on healthcare facilities globally due to the recent emergence of novel coronavirus infection known as COVID-19 and its rapid spread across the continents. The lack of effective therapeutics for the management of the pandemic calls for the discovery of new drugs and vaccines. OBJECTIVE: In the present study, a chemical library was screened for molecules against three coronavirus 3CL-like protease enzymes (SARS-CoV-2 3CLpro, SARS-CoV 3CLpro and MERS-CoV 3CLpro), which are a key player in the viral replication cycle. METHODS: Extensive computational methods such as virtual screening and molecular docking were employed in this study. RESULTS: Two lead molecules, ZINC08825480 (4-bromo-N'-{(E)-[1-phenyl-3-(pyridin-3-yl)-1H-pyrazol- 4-yl]methylidene}benzene-1-sulfonohydrazide) and ZINC72009942 (N-[[2-[[(3S)-3-methyl-1-piperidyl] methyl]phenyl]methyl]-6-oxo-1-(p-tolyl)-4,5-dihydro-1,2,4-triazine-3-carboxamide), were identified with better affinity with the three target enzymes as compared to the approved antiviral drugs. Both the lead molecules possessed favorable drug-like properties, fit well into the active site pocket close to His- Cys dyad and showed a good number of hydrogen bonds with the backbone as well as side chains of key amino acid residues. CONCLUSION: Thus, the present study offers two novel chemical entities against coronavirus infections which can be validated through various biological assays.