Antiviral peptides inhibiting the main protease of SARS-CoV-2 investigated by computational screening and in vitro protease assay.

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in viral replication and transcription and received great attention as a vital target for drug/peptide development. Therapeutic agents such as small-molecule drugs or peptides that interact with the Cys-His present in the catalytic site of Mpro are an efficient way to inhibit the protease. Although several emergency-approved vaccines showed good efficacy and drastically dropped the infection rate, evolving variants are still infecting and killing millions of people globally. While a small-molecule drug (Paxlovid) received emergency approval, small-molecule drugs have low target specificity and higher toxicity. Besides small-molecule drugs, peptide therapeutics are thus gaining increasing popularity as they are easy to synthesize and highly selective and have limited side effects. In this study, we investigated the therapeutic value of 67 peptides targeting Mpro using molecular docking. Subsequently, molecular dynamics (MD) simulations were implemented on eight protein-peptide complexes to obtain molecular-level information on the interaction between these peptides and the Mpro active site, which revealed that temporin L, indolicidin, and lymphocytic choriomeningitis virus (LCMV) GP1 are the best candidates in terms of stability, interaction, and structural compactness. These peptides were synthesized using the solid-phase peptide synthesis protocol, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and authenticated by mass spectrometry (MS). The in vitro fluorometric Mpro activity assay was used to validate the computational results, where temporin L and indolicidin were observed to be very active against SARS-CoV-2 Mpro with IC50 values of 38.80 and 87.23 µM, respectively. A liquid chromatography-MS (LC-MS) assay was developed, and the IC50 value of temporin L was measured at 23.8 µM. The solution-state nuclear magnetic resonance (NMR) structure of temporin L was determined in the absence of sodium dodecyl sulfate (SDS) micelles and was compared to previous temporin structures. This combined investigation provides critical insights and assists us to further develop peptide inhibitors of SARS-CoV-2 Mpro through structural guided investigation.

Potential antiviral peptides against the nucleoprotein of SARS-CoV-2.

Nucleoprotein is a conserved structural protein of SARS-CoV-2, which is involved in several functions, including replication, packaging, and transcription. In this research, 21 antiviral peptides that are known to have inhibitory function against nucleoprotein in several other viruses, were screened computationally against the nucleoprotein of SARS-CoV-2. The complexes of five best performing peptides (AVP1142, AVP1145, AVP1148, AVP1150, AVP1155) with nucleoprotein were selected for subsequent screening via 5 ns molecular dynamics (MD) simulation. Two peptides, namely AVP1145 and AVP1155, came out as promising candidates and hence were selected for 200 ns MD simulation for further validation, incorporating a DMPC-based membrane environment. In the long MD simulation, both AVP1155 and AVP1145 utilized multiple residues-mainly aromatic, acidic, and nonpolar residues-as interacting points to remain in contact with the nucleoprotein and formed predominantly hydrogen bonds along with hydrophobic and electrostatic interactions. However, AVP1155 proved to be superior to AVP1145 when its complex with nucleoprotein was analyzed in terms of root-mean-square deviation, root-mean-square fluctuation, radius of gyration, solvent accessible surface area and free energy landscape. In a nutshell, the findings of this research may guide future studies in the development of selective peptide inhibitors of SARS-CoV-2 nucleoprotein. Supplementary Information: The online version contains supplementary material available at 10.1007/s11696-022-02514-4.

Cysteine focused covalent inhibitors against the main protease of SARS-CoV-2.

In viral replication and transcription, the main protease (Mpro) of SARS-CoV-2 plays an important role and appears to be a vital target for drug design. In Mpro, there is a Cys-His catalytic dyad, and ligands that interact with the Cys145 assumed to be an effective approach to inhibit the Mpro. In this study, approximately 1400 cysteine-focused ligands were screened to identify the best candidates that can act as potent inhibitors against Mpro. Our results show that the selected ligands strongly interact with the key Cys145 and His41 residues. Covalent docking was performed for the selected candidates containing the acrylonitrile group, which can form a covalent bond with Cys145. All atoms molecular dynamics (MD) simulation was performed on the selected four inhibitors including L1, L2, L3 and L4 to validate the docking interactions. Our results were also compared with a control ligand, α-ketoamide (11r). Principal component analysis on structural and energy data obtained from the MD trajectories shows that L1, L3, L4 and α-ketoamide (11r) have structural similarity with the apo-form of the Mpro. Quantitative structure-activity relationship method was employed for pattern recognition of the best ligands, which discloses that ligands containing acrylonitrile and amide warheads can show better performance. ADMET analysis displays that our selected candidates appear to be safer inhibitors. Our combined studies suggest that the best cysteine focused ligands can help to design an effective lead drug for COVID-19 treatment. Communicated by Ramaswamy H. Sarma.

Computational screening of 645 antiviral peptides against the receptor-binding domain of the spike protein in SARS-CoV-2.

The receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein plays a vital role in binding and internalization through the alpha-helix (AH) of human angiotensin-converting enzyme 2 (hACE2). Thus, it is a potential target for designing and developing antiviral agents. Inhibition of RBD activity of the S protein may be achieved by blocking RBD interaction with hACE2. In this context, inhibitors with large contact surface area are preferable as they can form a potentially stable complex with RBD of S protein and would not allow RBD to come in contact with hACE2. Peptides represent excellent features as potential anti-RBD agents due to better efficacy, safety, and tolerability in humans compared to that of small molecules. The present study has selected 645 antiviral peptides known to inhibit various viruses and computationally screened them against the RBD of SARS-CoV-2 S protein. In primary screening, 27 out of 645 peptides exhibited higher affinity for the RBD of S protein compared to that of AH of the hACE2 receptor. Subsequently, AVP1795 appeared as the most promising candidate that could inhibit hACE2 recognition by SARS-CoV 2 as was predicted by the molecular dynamics simulation. The critical residues in RBD found for protein-peptide interactions are TYR 489, GLY 485, TYR 505, and GLU 484. Peptide-protein interactions were substantially influenced by hydrogen bonding and hydrophobic interactions. This comprehensive computational screening may provide a guideline to design the most effective peptides targeting the spike protein, which could be studied further in vitro and in vivo for assessing their anti-SARS CoV-2 activity.

Investigating the binding affinity, interaction, and structure-activity-relationship of 76 prescription antiviral drugs targeting RdRp and Mpro of SARS-CoV-2.

SARS-CoV-2 virus outbreak poses a major threat to humans worldwide due to its highly contagious nature. In this study, molecular docking, molecular dynamics, and structure-activity relationship are employed to assess the binding affinity and interaction of 76 prescription drugs against RNA dependent RNA polymerase (RdRp) and Main Protease (Mpro) of SARS-CoV-2. The RNA-dependent RNA polymerase is a vital enzyme of coronavirus replication/transcription complex whereas the main protease acts on the proteolysis of replicase polyproteins. Among 76 prescription antiviral drugs, four drugs (Raltegravir, Simeprevir, Cobicistat, and Daclatasvir) that are previously used for human immunodeficiency virus (HIV), hepatitis C virus (HCV), Ebola, and Marburg virus show higher binding energy and strong interaction with active sites of the receptor proteins. To explore the dynamic nature of the interaction, 100 ns molecular dynamics (MD) simulation is performed on the selected protein-drug complexes and apo-protein. Binding free energy of the selected drugs is performed by MM/PBSA. Besides docking and dynamics, partial least square (PLS) regression method is applied for the quantitative structure activity relationship to generate and predict the binding energy for drugs. PLS regression satisfactorily predicts the binding energy of the effective antiviral drugs compared to binding energy achieved from molecular docking with a precision of 85%. This study highly recommends researchers to screen these potential drugs in vitro and in vivo against SARS-CoV-2 for further validation of utility.

Virtual screening, molecular dynamics and structure-activity relationship studies to identify potent approved drugs for Covid-19 treatment.

Computer-aided drug screening by molecular docking, molecular dynamics (MD) and structural-activity relationship (SAR) can offer an efficient approach to identify promising drug repurposing candidates for COVID-19 treatment. In this study, computational screening is performed by molecular docking of 1615 Food and Drug Administration (FDA) approved drugs against the main protease (Mpro) of SARS-CoV-2. Several promising approved drugs, including Simeprevir, Ergotamine, Bromocriptine and Tadalafil, stand out as the best candidates based on their binding energy, fitting score and noncovalent interactions at the binding sites of the receptor. All selected drugs interact with the key active site residues, including His41 and Cys145. Various noncovalent interactions including hydrogen bonding, hydrophobic interactions, pi-sulfur and pi-pi interactions appear to be dominant in drug-Mpro complexes. MD simulations are applied for the most promising drugs. Structural stability and compactness are observed for the drug-Mpro complexes. The protein shows low flexibility in both apo and holo form during MD simulations. The MM/PBSA binding free energies are also measured for the selected drugs. For pattern recognition, structural similarity and binding energy prediction, multiple linear regression (MLR) models are used for the quantitative structural-activity relationship. The binding energy predicted by MLR model shows an 82% accuracy with the binding energy determined by molecular docking. Our details results can facilitate rational drug design targeting the SARS-CoV-2 main protease.Communicated by Ramaswamy H. Sarma.

A molecular modeling approach to identify effective antiviral phytochemicals against the main protease of SARS-CoV-2.

The main protease of SARS-CoV-2 is one of the important targets to design and develop antiviral drugs. In this study, we have selected 40 antiviral phytochemicals to find out the best candidates which can act as potent inhibitors against the main protease. Molecular docking is performed using AutoDock Vina and GOLD suite to determine the binding affinities and interactions between the phytochemicals and the main protease. The selected candidates strongly interact with the key Cys145 and His41 residues. To validate the docking interactions, 100 ns molecular dynamics (MD) simulations on the five top-ranked inhibitors including hypericin, cyanidin 3-glucoside, baicalin, glabridin, and α-ketoamide-11r are performed. Principal component analysis (PCA) on the MD simulation discloses that baicalin, cyanidin 3-glucoside, and α-ketoamide-11r have structural similarity with the apo-form of the main protease. These findings are also strongly supported by root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA) investigations. PCA is also used to find out the quantitative structure-activity relationship (QSAR) for pattern recognition of the best ligands. Multiple linear regression (MLR) of QSAR reveals the R2 value of 0.842 for the training set and 0.753 for the test set. Our proposed MLR model can predict the favorable binding energy compared with the binding energy detected from molecular docking. ADMET analysis demonstrates that these candidates appear to be safer inhibitors. Our comprehensive computational and statistical analysis show that these selected phytochemicals can be used as potential inhibitors against the SARS-CoV-2.Communicated by Ramaswamy H. Sarma.

Antiviral Peptides as Promising Therapeutics against SARS-CoV-2.

Over 50 peptides, which were known to inhibit SARS-CoV-1, were computationally screened against the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. Based on the binding affinity and interaction, 15 peptides were selected, which showed higher affinity compared to the α-helix of the human ACE2 receptor. Molecular dynamics simulation demonstrated that two peptides, S2P25 and S2P26, were the most promising candidates, which could potentially block the entry of SARS-CoV-2. Tyr489 and Tyr505 residues present in the "finger-like" projections of the RBD were found to be critical for peptide interaction. Hydrogen bonding and hydrophobic interactions played important roles in prompting peptide-protein binding and interaction. Structure-activity relationship indicated that peptides containing aromatic (Tyr and Phe), nonpolar (Pro, Gly, Leu, and Ala), and polar (Asn, Gln, and Cys) residues were the most significant contributors. These findings can facilitate the rational design of selective peptide inhibitors targeting the spike protein of SARS-CoV-2.