A Potential Peptide From Soy Cheese Produced Using Lactobacillus delbrueckii WS4 for Effective Inhibition of SARS-CoV-2 Main Protease and S1 Glycoprotein.
ABSTRACT: The COVID-19 pandemic caused by novel SARS-CoV-2 has resulted in an unprecedented loss of lives and economy around the world. In this study, search for potential inhibitors against two of the best characterized SARS-CoV-2 drug targets: S1 glycoprotein receptor-binding domain (RBD) and main protease (3CLPro), was carried out using the soy cheese peptides. A total of 1,420 peptides identified from the cheese peptidome produced using Lactobacillus delbrueckii WS4 were screened for antiviral activity by employing the web tools, AVPpred, and meta-iAVP. Molecular docking studies of the selected peptides revealed one potential peptide "KFVPKQPNMIL" that demonstrated strong affinity toward significant amino acid residues responsible for the host cell entry (RBD) and multiplication (3CLpro) of SARS-CoV-2. The peptide was also assessed for its ability to interact with the critical residues of S1 RBD and 3CLpro of other ?-coronaviruses. High binding affinity was observed toward critical amino acids of both the targeted proteins in SARS-CoV, MERS-CoV, and HCoV-HKU1. The binding energy of KFVPKQPNMIL against RBD and 3CLpro of the four viruses ranged from -8.45 to -26.8 kcal/mol and -15.22 to -22.85 kcal/mol, respectively. The findings conclude that cheese, produced by using Lb. delbrueckii WS4, could be explored as a prophylactic food for SARS-CoV-2 and related viruses. In addition, the multi-target inhibitor peptide, which effectively inhibited both the viral proteins, could further be used as a terminus a quo for the in vitro and in vivo function against SARS-CoV-2.
Project description:The 3C-like protease (3CL<sup>pro</sup>) of SARS-CoV-2 is considered an excellent target for COVID-19 antiviral drug development because it is essential for viral replication and has a cleavage specificity distinct from human proteases. However, drug development for 3CL<sup>pro</sup> has been hindered by a lack of cell-based reporter assays that can be performed in a BSL-2 setting. Current efforts to identify 3CL<sup>pro</sup> inhibitors largely rely upon in vitro screening, which fails to account for cell permeability and cytotoxicity of compounds, or assays involving replication-competent virus, which must be performed in a BSL-3 facility. To address these limitations, we have developed a novel cell-based luciferase complementation reporter assay to identify inhibitors of SARS-CoV-2 3CL<sup>pro</sup> in a BSL-2 setting. The assay is based on a lentiviral vector that co-expresses 3CL<sup>pro</sup> and two luciferase fragments linked together by a 3CL<sup>pro</sup> cleavage site. 3CL<sup>pro</sup>-mediated cleavage results in a loss of complementation and low luciferase activity, whereas inhibition of 3CL<sup>pro</sup> results in 10-fold higher levels of luciferase activity. The luciferase reporter assay can easily distinguish true 3CL<sup>pro</sup> inhibition from cytotoxicity, a powerful feature that should reduce false positives during screening. Using the assay, we screened 32 small molecules for activity against SARS-CoV-2 3CL<sup>pro</sup>, including HIV protease inhibitors, HCV protease inhibitors, and various other compounds that have been reported to inhibit SARS-CoV-2 3CL<sup>pro</sup>. Of these, only five exhibited significant inhibition of 3CL<sup>pro</sup> in cells: GC376, boceprevir, Z-FA-FMK, calpain inhibitor XII, and GRL-0496. This assay should greatly facilitate efforts to identify more potent inhibitors of SARS-CoV-2 3CL<sup>pro</sup>.
Project description:COVID-19 has become a global pandemic and there is an urgent call for developing drugs against the virus (SARS-CoV-2). The 3C-like protease (3CL<sup>pro</sup>) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of <i>S. baicalensis</i> and its ingredients. We found that the ethanol extract of <i>S. baicalensis</i> and its major component, baicalein, inhibit SARS-CoV-2 3CL<sup>pro</sup> activity <i>in vitro</i> with IC<sub>50</sub>'s of 8.52 µg/ml and 0.39 µM, respectively. Both of them inhibit the replication of SARS-CoV-2 in Vero cells with EC<sub>50</sub>'s of 0.74 µg/ml and 2.9 µM, respectively. While baicalein is mainly active at the viral post-entry stage, the ethanol extract also inhibits viral entry. We further identified four baicalein analogues from other herbs that inhibit SARS-CoV-2 3CL<sup>pro</sup> activity at µM concentration. All the active compounds and the <i>S. baicalensis</i> extract also inhibit the SARS-CoV 3CL<sup>pro</sup>, demonstrating their potential as broad-spectrum anti-coronavirus drugs.
Project description:<h4>Background</h4>In the urgent campaign to develop therapeutics against SARS-CoV-2, natural products have been an important source of new lead compounds.<h4>Results</h4>We herein identified two natural products, ginkgolic acid and anacardic acid, as inhibitors using a high-throughput screen targeting the SARS-CoV-2 papain-like protease (PL<sup>pro</sup>). Moreover, our study demonstrated that the two hit compounds are dual inhibitors targeting the SARS-CoV-2 3-chymotrypsin-like protease (3CL<sup>pro</sup>) in addition to PL<sup>pro</sup>. A mechanism of action study using enzyme kinetics further characterized the two compounds as irreversible inhibitors against both 3CL<sup>pro</sup> and PL<sup>pro</sup>. Significantly, both identified compounds inhibit SARS-CoV-2 replication in vitro at nontoxic concentrations.<h4>Conclusions</h4>Our finding provides two novel natural products as promising SARS-CoV-2 antivirals.
Project description:<h4>Background</h4>Since the first patient identified with SARS-CoV-2 symptoms in December 2019, the trend of a spreading coronavirus disease 2019 (COVID-19) infection has remained to date. As for now, there is an urgent need to develop novel drugs or vaccines for the SARS-CoV-2 virus.<h4>Methods</h4>Polyphenolic compounds have potential as drug candidates for various diseases, including viral infections. In this study, polyphenolic compounds contained in Geranii Herba were chosen for an in silico approach. The SARS-CoV-2 receptor-binding domain (RBD), 3CL<sup>pro</sup> (Replicase polyprotein 1ab), and the cell surface receptor glucose-regulated protein 78 (GRP78) were chosen as target proteins.<h4>Results</h4>Based on the molecular docking analysis, ellagic acid, gallic acid, geraniin, kaempferitrin, kaempferol, and quercetin showed significant binding interactions with the target proteins. Besides, the molecular dynamic simulation studies support Geranii Herba's inhibition efficiency on the SARS-CoV-2 RBD. We assume that the active compounds in Geranii Herba might inhibit SARS-CoV-2 cell entry through the ACE2 receptor and inhibit the proteolytic process. Besides, these compounds may help to regulate the cell signaling under the unfolded protein response in endoplasmic reticulum stress through the binding with GRP78 and avoid the SARS-CoV-2 interaction.<h4>Conclusions</h4>Hence, the compounds present in Geranii Herba could be used as possible drug candidates for the prevention/treatment of SARS-CoV-2 infection.
Project description:3CL proteases (3CL<sup>pro</sup>) are only found in RNA viruses and have a central role in polyprotein processing during replication. Therefore, 3CL<sup>pro</sup> has emerged as promising drug target for therapeutic treatment of infections caused by Coronaviruses. In the light of the recent major outbreak of the SARS-CoV-2 virus and the continuously rising numbers of infections and casualties, there is an urgent need for quickly available drugs or vaccines to stop the current COVID-19 pandemic. Repurposing of approved drugs as 3CL<sup>pro</sup> inhibitors could dramatically shorten the period up to approval as therapeutic against SARS-CoV-2, since pharmacokinetics and toxicity is already known. Several known drugs, e.g. oxytetracycline, doxorubicin, kanamycin, cefpiramide, teniposide, proanthocyanidin and salvianolic acid B, but also not-approved active compounds from the ZINC15 library were identified as new potential inhibitors of 3CL<sup>pro</sup> by using different complementary virtual screening and docking approaches. These compounds have the potential to be further optimized using structure based drug design as demonstrated for oxytetracycline.
Project description:With the rapid growth of the COVID-19 (coronavirus disease 2019) pandemic across the globe, therapeutic attention must be directed to fight the novel severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). However, developing new antiviral drugs and vaccines is time-consuming, so one of the best solutions to tackle this virus at present is to repurpose ready-to-use drugs. This paper proposes the repurposing of the Food and Drug Administration (FDA)-approved, purchasable, and naturally occurring drugs for preventive and therapeutic use. We propose to design a dual-inhibitor for the SARS-CoV-2 cysteine proteases-3 Chemotrypsin-like protease or main protease (3CL<sup>pro</sup> or M<sup>pro</sup>) and Papain-like protease (PL<sup>pro</sup>) responsible for processing the translated polyprotein chain from the viral RNA yielding functional viral proteins. For virtual screening, an unbiased blind docking was performed from which the top nine dual-targeting inhibitors for 3CL<sup>pro</sup> and PL<sup>pro</sup> were selected. The nine repurposed drugs, block the catalytic dyad (His41 and Cys145) of 3CL<sup>pro</sup> as well as the catalytic triad (Cys111, His272, and Asp286) of PL<sup>pro</sup>. Repurposing known drugs will not only pave the way for rapid in-vitro and in-vivo studies to battle the SARS-CoV-2 but will also expedite the quest for a potent anti-coronaviral drug.
Project description:The emergence outbreak caused by a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has received significant attention on the global risks. Due to itscrucial role in viral replication, the main protease 3CL<sup>pro</sup> is an important target for drug discovery and development to combat COVID-19. In this work, the structural and dynamic behaviors as well as binding efficiency of the four peptidomimetic inhibitors (N3, 11a, 13b, and 14b) recently co-crystalized with SARS-CoV-2 3CL<sup>pro</sup> were studied and compared using all-atom molecular dynamics (MD) simulations and solvated interaction energy-based binding free energy calculations. The per-residue decomposition free energy results suggested that the key residues involved in inhibitors binding were H41, M49, L141-C145, H163-E166, P168, and Q189-T190 in the domains I and II. The van der Waals interaction yielded the main energy contribution stabilizing all the focused inhibitors. Besides, their hydrogen bond formations with F140, G143, C145, H164, E166, and Q189 residues in the substrate-binding pocket were also essential for strengthening the molecular complexation. The predicted binding affinity of the four peptidomimetic inhibitors agreed with the reported experimental data, and the 13b showed the most efficient binding to SARS-CoV-2 3CL<sup>pro</sup>. From rational drug design strategies based on 13b, the polar moieties (e.g., benzamide) and the bulky N-terminal protecting groups (e.g., thiazole) should be introduced to P1' and P4 sites in order to enhance H-bonds and hydrophobic interactions, respectively. We hope that the obtained structural and energetic information could be beneficial for developing novel SARS-CoV-2 3CL<sup>pro</sup> inhibitors with higher inhibitory potency to combat COVID-19.
Project description:Bioflavonoids are the largest group of plant-derived polyphenolic compounds with diverse biological potential and have also been proven efficacious in the treatment of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). The present investigation validates molecular docking, simulation, and MM-PBSA studies of fifteen bioactive bioflavonoids derived from plants as a plausible potential antiviral in the treatment of COVID-19. Molecular docking studies for 15 flavonoids on the three SARS CoV-2 proteins, non-structural protein-15 Endoribonuclease (NSP15), the receptor-binding domain of spike protein (RBD of S protein), and main protease (M<sup>pro</sup>/3CL<sup>pro</sup>) were performed and selected protein-ligand complexes were subjected to Molecular Dynamics simulations. The molecular dynamics trajectories were subjected to free energy calculation by the MM-PBSA method. All flavonoids were further assessed for their effectiveness as adjuvant therapy by network pharmacology analysis on the target proteins. The network pharmacology analysis suggests the involvement of selected bioflavonoids in the modulation of multiple signaling pathways like p53, FoxO, MAPK, Wnt, Rap1, TNF, adipocytokine, and leukocyte transendothelial migration which plays a significant role in immunomodulation, minimizing the oxidative stress and inflammation. Molecular docking and molecular dynamics simulation studies illustrated the potential of glycyrrhizic acid, amentoflavone, and mulberroside in inhibiting key SARS-CoV-2 proteins and these results could be exploited further in designing future ligands from natural sources.
Project description:Severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CL(pro)) mediates extensive proteolytic processing of replicase polyproteins, and is considered a promising target for anti-SARS drug development. Here we present a rapid and high-throughput screening method to study the substrate specificity of SARS-CoV 3CL(pro). Six target amino acid positions flanking the SARS-CoV 3CL(pro) cleavage site were investigated. Each batch of mixed peptide substrates with defined amino acid substitutions at the target amino acid position was synthesized via the "cartridge replacement" approach and was subjected to enzymatic cleavage by recombinant SARS-CoV 3CL(pro). Susceptibility of each peptide substrate to SARS-CoV 3CL(pro) cleavage was monitored simultaneously by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The hydrophobic pocket in the P2 position at the protease cleavage site is crucial to SARS-CoV 3CL(pro)-specific binding, which is limited to substitution by hydrophobic residue. The binding interface of SARS-CoV 3CL(pro) that is facing the P1' position is suggested to be occupied by acidic amino acids, thus the P1' position is intolerant to acidic residue substitution, owing to electrostatic repulsion. Steric hindrance caused by some bulky or beta-branching amino acids in P3 and P2' positions may also hinder the binding of SARS-CoV 3CL(pro). This study generates a comprehensive overview of SARS-CoV 3CL(pro) substrate specificity, which serves as the design basis of synthetic peptide-based SARS-CoV 3CL(pro) inhibitors. Our experimental approach is believed to be widely applicable for investigating the substrate specificity of other proteases in a rapid and high-throughput manner that is compatible for future automated analysis.
Project description:The COVID-19 pandemic caused by SARS-CoV-2 requires rapid development of specific therapeutics and vaccines. The main protease of SARS-CoV-2, 3CL M<sup>pro</sup>, is an established drug target for the design of inhibitors to stop the virus replication. Repurposing existing clinical drugs can offer a faster route to treatments. Here, we report on the binding mode and inhibition properties of several inhibitors using room temperature X-ray crystallography and in vitro enzyme kinetics. The enzyme active-site cavity reveals a high degree of malleability, allowing aldehyde leupeptin and hepatitis C clinical protease inhibitors (telaprevir, narlaprevir, and boceprevir) to bind and inhibit SARS-CoV-2 3CL M<sup>pro</sup>. Narlaprevir, boceprevir, and telaprevir are low-micromolar inhibitors, whereas the binding affinity of leupeptin is substantially weaker. Repurposing hepatitis C clinical drugs as COVID-19 treatments may be a useful option to pursue. The observed malleability of the enzyme active-site cavity should be considered for the successful design of specific protease inhibitors.