Non-Toxic Dimeric Peptides Derived from the Bothropstoxin-I Are Potent SARS-CoV-2 and Papain-like Protease Inhibitors

The COVID-19 outbreak has rapidly spread on a global scale, affecting the economy and public health systems throughout the world. In recent years, peptide-based therapeutics have been widely studied and developed to treat infectious diseases, including viral infections. Herein, the antiviral effects of the lysine linked dimer des-Cys¹¹, Lys¹²,Lys¹³-(pBthTX-I)₂K ((pBthTX-I)₂K)) and derivatives against SARS-CoV-2 are reported. The lead peptide (pBthTX-I)₂K and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC₅₀ = 28–65 µM) and mostly low cytotoxic effect (CC₅₀ > 100 µM). To shed light on the mechanism of action underlying the peptides’ antiviral activity, the Main Protease (M^pro) and Papain-Like protease (PL^pro) inhibitory activities of the peptides were assessed. The synthetic peptides showed PL^pro inhibition potencies (IC₅₀s = 1.0–3.5 µM) and binding affinities (K_d = 0.9–7 µM) at the low micromolar range but poor inhibitory activity against M^pro (IC₅₀ > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PL^pro substrate toward the protease catalytic cleft. Our findings indicated that non-toxic dimeric peptides derived from the Bothropstoxin-I have attractive cellular and enzymatic inhibitory activities, thereby suggesting that they are promising prototypes for the discovery and development of new drugs against SARS-CoV-2 infection.     

Computational Simulation of HIV Protease Inhibitors to the Main Protease (Mpro) of SARS-CoV-2: Implications for COVID-19 Drugs Design

SARS-CoV-2 is highly homologous to SARS-CoV. To date, the main protease (Mpro) of SARS-CoV-2 is regarded as an important drug target for the treatment of Coronavirus Disease 2019 (COVID-19). Some experiments confirmed that several HIV protease inhibitors present the inhibitory effects on the replication of SARS-CoV-2 by inhibiting Mpro. However, the mechanism of action has still not been studied very clearly. In this work, the interaction mechanism of four HIV protease inhibitors Darunavir (DRV), Lopinavir (LPV), Nelfinavir (NFV), and Ritonavire (RTV) targeting SARS-CoV-2 Mpro was explored by applying docking, molecular dynamics (MD) simulations, and MM–GBSA methods using the broad-spectrum antiviral drug Ribavirin (RBV) as the negative and nonspecific control. Our results revealed that LPV, RTV, and NFV have higher binding affinities with Mpro, and they all interact with catalytic residues His41 and the other two key amino acids Met49 and Met165. Pharmacophore model analysis further revealed that the aromatic ring, hydrogen bond donor, and hydrophobic group are the essential infrastructure of Mpro inhibitors. Overall, this study applied computational simulation methods to study the interaction mechanism of HIV-1 protease inhibitors with SARS-CoV-2 Mpro, and the findings provide useful insights for the development of novel anti-SARS-CoV-2 agents for the treatment of COVID-19.     

SARS-CoV-2 Main Protease Active Site Ligands in the Human Metabolome

In late 2019, a global pandemic occurred. The causative agent was identified as a member of the Coronaviridae family, called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we present an analysis on the substances identified in the human metabolome capable of binding the active site of the SARS-CoV-2 main protease (M^pro). The substances present in the human metabolome have both endogenous and exogenous origins. The aim of this research was to find molecules whose biochemical and toxicological profile was known that could be the starting point for the development of antiviral therapies. Our analysis revealed numerous metabolites—including xenobiotics—that bind this protease, which are essential to the lifecycle of the virus. Among these substances, silybin, a flavolignan compound and the main active component of silymarin, is particularly noteworthy. Silymarin is a standardized extract of milk thistle, Silybum marianum, and has been shown to exhibit antioxidant, hepatoprotective, antineoplastic, and antiviral activities. Our results—obtained in silico and in vitro—prove that silybin and silymarin, respectively, are able to inhibit M^pro, representing a possible food-derived natural compound that is useful as a therapeutic strategy against COVID-19.     

Thiazole/Thiadiazole/Benzothiazole Based Thiazolidin-4-One Derivatives as Potential Inhibitors of Main Protease of SARS-CoV-2

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 IC₅₀ 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.     

Identification of Potential SARS-CoV-2 Main Protease and Spike Protein Inhibitors from the Genus Aloe: An In Silico Study for Drug Development

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) disease is a global rapidly spreading virus showing very high rates of complications and mortality. Till now, there is no effective specific treatment for the disease. Aloe is a rich source of isolated phytoconstituents that have an enormous range of biological activities. Since there are no available experimental techniques to examine these compounds for antiviral activity against SARS-CoV-2, we employed an in silico approach involving molecular docking, dynamics simulation, and binding free energy calculation using SARS-CoV-2 essential proteins as main protease and spike protein to identify lead compounds from Aloe that may help in novel drug discovery. Results retrieved from docking and molecular dynamics simulation suggested a number of promising inhibitors from Aloe. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) calculations indicated that compounds 132, 134, and 159 were the best scoring compounds against main protease, while compounds 115, 120, and 131 were the best scoring ones against spike glycoprotein. Compounds 120 and 131 were able to achieve significant stability and binding free energies during molecular dynamics simulation. In addition, the highest scoring compounds were investigated for their pharmacokinetic properties and drug-likeness. The Aloe compounds are promising active phytoconstituents for drug development for SARS-CoV-2.     

Phytochemical Discrimination,Biological Activity and Molecular Docking of Water-Soluble Inhibitors from Saussurea costus Herb against Main Protease of SARS-CoV-2

Siddha medicine is one of the oldest medical systems in the world and is believed to have originated more than 10,000 years ago and is prevalent across ancient Tamil land. It is undeniable that inhibitor preferences rise with increasing solubility in water due to the considerations pertaining to the bioavailability and the ease of which unabsorbed residues can be disposed of. In this study, we showed the phytochemical discrimination of Saussurea costus extracted with water at room temperature as a green extraction procedure. A total of 48 compounds were identified using gas chromatography-mass spectrometry (GC-MS). The fatty acids had a high phytochemical abundance at 73.8%, followed by tannins at 8.2%, carbohydrates at 6.9%, terpenoids at 4.3%, carboxylic acids at 2.5%, hydrocarbons at 2.4%, phenolic compounds at 0.2%, and sterols at 1.5%. Of these compounds, 22 were docked on the active side and on the catalytic dyad of His41 and Cys145 of the main protease of SARS-CoV-2 (M^pro). Eight active inhibitors were carbohydrates, five were fatty acids, three were terpenoids, two were carboxylic acids, one was a tannin, one was a phenolic compound, and one was a sterol. The best inhibitors were 4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl), Andrographolide, and delta.4-Androstene-3.beta.,17.beta.-diol, with a binding affinity that ranged from −6.1 kcal/mol to −6.5 kcal/mol. The inhibitory effect of Saussurea costus of SARS-CoV-2 entry into the cell was studied using a pseudovirus with Spike proteins from the D614G variant and the VOC variants Gamma and Delta. Based on the viral cycle of SARS-CoV-2, our results suggest that the Saussurea costus aqueous extract has no virucidal effect and inhibits the virus in the events after cell entry. Furthermore, the biological activity of the aqueous extract was investigated against HSV-1 virus and two bacterial strains, namely Staphylococcus aureus ATCC BAA 1026 and Escherichia coli ATCC 9637. According to this study, an enormous number of water-soluble inhibitors were identified from Saussurea costus against the M^pro, and this is unprecedented as far as we know.     

Triterpenic Acid Amides as Potential Inhibitors of the SARS-CoV-2 Main Protease

Although the incidence and mortality of SARS-CoV-2 infection has been declining during the pandemic, the problem related to designing novel antiviral drugs that could effectively resist viruses in the future remains relevant. As part of our continued search for chemical compounds that are capable of exerting an antiviral effect against the SARS-CoV-2 virus, we studied the ability of triterpenic acid amides to inhibit the SARS-CoV-2 main protease. Molecular modeling suggested that the compounds are able to bind to the active site of the main protease via non-covalent interactions. The FRET-based enzyme assay was used to reveal that compounds 1e and 1b can inhibit the SARS-CoV-2 main protease at micromolar concentrations.     

Molecular Docking and Dynamics Investigations for Identifying Potential Inhibitors of the 3-Chymotrypsin-like Protease of SARS-CoV-2: Repurposing of Approved Pyrimidonic Pharmaceuticals for COVID-19 Treatment

This study demonstrates the inhibitory effect of 42 pyrimidonic pharmaceuticals (PPs) on the 3-chymotrypsin-like protease of SARS-CoV-2 (3CL^pro) through molecular docking, molecular dynamics simulations, and free binding energies by means of molecular mechanics–Poisson Boltzmann surface area (MM-PBSA) and molecular mechanics–generalized Born surface area (MM-GBSA). Of these tested PPs, 11 drugs approved by the US Food and Drug Administration showed an excellent binding affinity to the catalytic residues of 3CL^pro of His41 and Cys145: uracil mustard, cytarabine, floxuridine, trifluridine, stavudine, lamivudine, zalcitabine, telbivudine, tipiracil, citicoline, and uridine triacetate. Their percentage of residues involved in binding at the active sites ranged from 56 to 100, and their binding affinities were in the range from −4.6 ± 0.14 to −7.0 ± 0.19 kcal/mol. The molecular dynamics as determined by a 200 ns simulation run of solvated docked complexes confirmed the stability of PP conformations that bound to the catalytic dyad and the active sites of 3CL^pro. The free energy of binding also demonstrates the stability of the PP–3CL^pro complexes. Citicoline and uridine triacetate showed free binding energies of −25.53 and −7.07 kcal/mol, respectively. Therefore, I recommend that they be repurposed for the fight against COVID-19, following proper experimental and clinical validation.     

In Silico Mining of Terpenes from Red-Sea Invertebrates for SARS-CoV-2 Main Protease (Mpro) Inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic, which generated more than 1.82 million deaths in 2020 alone, in addition to 83.8 million infections. Currently, there is no antiviral medication to treat COVID-19. In the search for drug leads, marine-derived metabolites are reported here as prospective SARS-CoV-2 inhibitors. Two hundred and twenty-seven terpene natural products isolated from the biodiverse Red-Sea ecosystem were screened for inhibitor activity against the SARS-CoV-2 main protease (M^pro) using molecular docking and molecular dynamics (MD) simulations combined with molecular mechanics/generalized Born surface area binding energy calculations. On the basis of in silico analyses, six terpenes demonstrated high potency as M^pro inhibitors with ΔG_binding ≤ −40.0 kcal/mol. The stability and binding affinity of the most potent metabolite, erylosides B, were compared to the human immunodeficiency virus protease inhibitor, lopinavir. Erylosides B showed greater binding affinity towards SARS-CoV-2 M^pro than lopinavir over 100 ns with ΔG_binding values of −51.9 vs. −33.6 kcal/mol, respectively. Protein–protein interactions indicate that erylosides B biochemical signaling shares gene components that mediate severe acute respiratory syndrome diseases, including the cytokine- and immune-signaling components BCL2L1, IL2, and PRKC. Pathway enrichment analysis and Boolean network modeling were performed towards a deep dissection and mining of the erylosides B target–function interactions. The current study identifies erylosides B as a promising anti-COVID-19 drug lead that warrants further in vitro and in vivo testing.     

Chloropyridinyl Esters of Nonsteroidal Anti-Inflammatory Agents and Related Derivatives as Potent SARS-CoV-2 3CL Protease Inhibitors

We report the design and synthesis of a series of new 5-chloropyridinyl esters of salicylic acid, ibuprofen, indomethacin, and related aromatic carboxylic acids for evaluation against SARS-CoV-2 3CL protease enzyme. These ester derivatives were synthesized using EDC in the presence of DMAP to provide various esters in good to excellent yields. Compounds are stable and purified by silica gel chromatography and characterized using ¹H-NMR, ¹³C-NMR, and mass spectral analysis. These synthetic derivatives were evaluated in our in vitro SARS-CoV-2 3CLpro inhibition assay using authentic SARS-CoV-2 3CLpro enzyme. Compounds were also evaluated in our in vitro antiviral assay using quantitative VeroE₆ cell-based assay with RNAqPCR. A number of compounds exhibited potent SARS-CoV-2 3CLpro inhibitory activity and antiviral activity. Compound 9a was the most potent inhibitor, with an enzyme IC₅₀ value of 160 nM. Compound 13b exhibited an enzyme IC₅₀ value of 4.9 µM. However, it exhibited a potent antiviral EC₅₀ value of 24 µM in VeroE6 cells. Remdesivir, an RdRp inhibitor, exhibited an antiviral EC₅₀ value of 2.4 µM in the same assay. We assessed the mode of inhibition using mass spectral analysis which suggested the formation of a covalent bond with the enzyme. To obtain molecular insight, we have created a model of compound 9a bound to SARS-CoV-2 3CLpro in the active site.     

Ensemble Docking Coupled to Linear Interaction Energy Calculations for Identification of Coronavirus Main Protease (3CLpro) Non-Covalent Small-Molecule Inhibitors

SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new strain of Coronaviridae. In the closing 2019 to early 2020 months, the virus caused a global pandemic of COVID-19 disease. We performed a virtual screening study in order to identify potential inhibitors of the SARS-CoV-2 main viral protease (3CL^pro or M^pro). For this purpose, we developed a novel approach using ensemble docking high-throughput virtual screening directly coupled with subsequent Linear Interaction Energy (LIE) calculations to maximize the conformational space sampling and to assess the binding affinity of identified inhibitors. A large database of small commercial compounds was prepared, and top-scoring hits were identified with two compounds singled out, namely 1-[(R)-2-(1,3-benzimidazol-2-yl)-1-pyrrolidinyl]-2-(4-methyl-1,4-diazepan-1-yl)-1-ethanone and [({(S)-1-[(1H-indol-2-yl)methyl]-3-pyrrolidinyl}methyl)amino](5-methyl-2H-pyrazol-3-yl)formaldehyde. Moreover, we obtained a favorable binding free energy of the identified compounds, and using contact analysis we confirmed their stable binding modes in the 3CL^pro active site. These compounds will facilitate further 3CL^pro inhibitor design.     

Solanaceae Family Phytochemicals as Inhibitors of 3C-Like Protease of SARS-CoV-2: An In Silico Analysis

COVID-19, caused by the coronavirus SARS-CoV-2, emerged in late December 2019 in Wuhan, China. As of 8 April 2022, the virus has caused a global pandemic, resulting in 494,587,638 infections leading to 6,170,283 deaths around the world. Although several vaccines have received emergency authorization from USA and UK drug authorities and two more in Russia and China, it is too early to comment on the prolonged effectiveness of the vaccines, their availability, and affordability for the developing countries of the world, and the daunting task to vaccinate 7 billion people of the world with two doses of the vaccine with additional booster doses. As a result, it is still worthwhile to search for drugs and several promising leads have been found, mainly through in silico studies. In this study, we have examined the binding energies of several alkaloids and anthocyanin derivatives from the Solanaceae family, a family which contains common consumable vegetables and fruit items such as eggplant, pepper, and tomatoes. Our study demonstrates that Solanaceae family alkaloids such as incanumine and solaradixine, as well as anthocyanins and anthocyanidins, have very high predicted binding energies for the 3C-like protease of SARS-CoV-2 (also known as Mpro). Since Mpro is vital for SARS-CoV-2 replication, the compounds merit potential for further antiviral research towards the objective of obtaining affordable drugs.     

Design of SARS-CoV-2 Main Protease Inhibitors Using Artificial Intelligence and Molecular Dynamic Simulations

Drug design is a time-consuming and cumbersome process due to the vast search space of drug-like molecules and the difficulty of investigating atomic and electronic interactions. The present paper proposes a computational drug design workflow that combines artificial intelligence (AI) methods, i.e., an evolutionary algorithm and artificial neural network model, and molecular dynamics (MD) simulations to design and evaluate potential drug candidates. For the purpose of illustration, the proposed workflow was applied to design drug candidates against the main protease of severe acute respiratory syndrome coronavirus 2. From the ∼140,000 molecules designed using AI methods, MD analysis identified two molecules as potential drug candidates.     

In Silico Identification and Validation of Organic Triazole Based Ligands as Potential Inhibitory Drug Compounds of SARS-CoV-2 Main Protease

The SARS-CoV-2 virus is highly contagious to humans and has caused a pandemic of global proportions. Despite worldwide research efforts, efficient targeted therapies against the virus are still lacking. With the ready availability of the macromolecular structures of coronavirus and its known variants, the search for anti-SARS-CoV-2 therapeutics through in silico analysis has become a highly promising field of research. In this study, we investigate the inhibiting potentialities of triazole-based compounds against the SARS-CoV-2 main protease (M^pro). The SARS-CoV-2 main protease (M^pro) is known to play a prominent role in the processing of polyproteins that are translated from the viral RNA. Compounds were pre-screened from 171 candidates (collected from the DrugBank database). The results showed that four candidates (Bemcentinib, Bisoctrizole, PYIITM, and NIPFC) had high binding affinity values and had the potential to interrupt the main protease (M^pro) activities of the SARS-CoV-2 virus. The pharmacokinetic parameters of these candidates were assessed and through molecular dynamic (MD) simulation their stability, interaction, and conformation were analyzed. In summary, this study identified the most suitable compounds for targeting Mpro, and we recommend using these compounds as potential drug molecules against SARS-CoV-2 after follow up studies.     

Computational Screening of Phenylamino-Phenoxy-Quinoline Derivatives against the Main Protease of SARS-CoV-2 Using Molecular Docking and the ONIOM Method

In the search for new anti-HIV-1 agents, two forms of phenylamino-phenoxy-quinoline derivatives have been synthesized, namely, 2-phenylamino-4-phenoxy-quinoline and 6-phenylamino-4-phenoxy-quinoline. In this study, the binding interactions of phenylamino-phenoxy-quinoline derivatives and six commercially available drugs (hydroxychloroquine, ritonavir, remdesivir, S-217622, N3, and PF-07321332) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M^pro) were investigated using molecular docking and the ONIOM method. The molecular docking showed the hydrogen bonding and hydrophobic interactions of all the compounds in the pocket of SARS-CoV-2 main protease (M^pro), which plays an important role for the division and proliferation of the virus into the cell. The binding free energy values between the ligands and M^pro ranged from −7.06 to −10.61 kcal/mol. The molecular docking and ONIOM results suggested that 4-(2′,6′-dimethyl-4′-cyanophenoxy)-2-(4″-cyanophenyl)-aminoquinoline and 4-(4′-cyanophenoxy)-2-(4″-cyanophenyl)-aminoquinoline have low binding energy values and appropriate molecular properties; moreover, both compounds could bind to M^pro via hydrogen bonding and Pi-Pi stacking interactions with amino acid residues, namely, HIS41, GLU166, and GLN192. These amino acids are related to the proteolytic cleavage process of the catalytic triad mechanisms. Therefore, this study provides important information for further studies on synthetic quinoline derivatives as antiviral candidates in the treatment of SARS-CoV-2.     

Identification of SARS-CoV-2 Papain-like Protease (PLpro) Inhibitors Using Combined Computational Approach**

In the current pandemic, finding an effective drug to prevent or treat the infection is the highest priority. A rapid and safe approach to counteract COVID-19 is in silico drug repurposing. The SARS-CoV-2 PLpro promotes viral replication and modulates the host immune system, resulting in inhibition of the host antiviral innate immune response, and therefore is an attractive drug target. In this study, we used a combined in silico virtual screening for candidates for SARS-CoV-2 PLpro protease inhibitors. We used the Informational spectrum method applied for Small Molecules for searching the Drugbank database followed by molecular docking. After in silico screening of drug space, we identified 44 drugs as potential SARS-CoV-2 PLpro inhibitors that we propose for further experimental testing.     

Identification of SARS-CoV-2 Main Protease Inhibitors from a Library of Minor Cannabinoids by Biochemical Inhibition Assay and Surface Plasmon Resonance Characterized Binding Affinity

The replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its main protease (M^pro), which is a plausible therapeutic target for coronavirus disease 2019 (COVID-19). Although numerous in silico studies reported the potential inhibitory effects of natural products including cannabis and cannabinoids on SARS-CoV-2 M^pro, their anti-M^pro activities are not well validated by biological experimental data. Herein, a library of minor cannabinoids belonging to several chemotypes including tetrahydrocannabinols, cannabidiols, cannabigerols, cannabichromenes, cannabinodiols, cannabicyclols, cannabinols, and cannabitriols was evaluated for their anti-M^pro activity using a biochemical assay. Additionally, the binding affinities and molecular interactions between the active cannabinoids and the M^pro protein were studied by a biophysical technique (surface plasmon resonance; SPR) and molecular docking, respectively. Cannabinoids tetrahydrocannabutol and cannabigerolic acid were the most active M^pro inhibitors (IC₅₀ = 3.62 and 14.40 μM, respectively) and cannabigerolic acid had a binding affinity KD=2.16×10−4 M). A preliminary structure and activity relationship study revealed that the anti-Mpro effects of cannabinoids were influenced by the decarboxylation of cannabinoids and the length of cannabinoids’ alkyl side chain. Findings from the biochemical, biophysical, and computational assays support the growing evidence of cannabinoids’ inhibitory effects on SARS-CoV-2 M^pro.     

Colorimetric and Electrochemical Methods for the Detection of SARS-CoV-2 Main Protease by Peptide-Triggered Assembly of Gold Nanoparticles

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.