Virtual high throughput screening: Potential Inhibitors for SARS-CoV-2 PLPRO and 3CLPRO Proteases.
ABSTRACT: The pandemic, COVID-19, has spread worldwide and affected millions of people. There is an urgent need, therefore, to find a proper treatment for the novel coronavirus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), the causative agent. This paper focuses on identifying inhibitors that target SARS-CoV-2 proteases, PLPRO and 3CLPRO, which control the duplication and manages the life cycle of SARS-CoV-2. We have carried out detailed in silico Virtual high-throughput screening using Food and Drug Administration (FDA) approved drugs from the Zinc database, COVID-19 clinical trial compounds from Pubchem database, Natural compounds from Natural Product Activity and Species Source (NPASS) database and Maybridge database against PLPRO and 3CLPRO proteases. After thoroughly analyzing the screening results, we found five compounds, Bemcentinib, Pacritinib, Ergotamine, MFCD00832476, and MFCD02180753 inhibit PLPRO and six compounds, Bemcentinib, Clofazimine, Abivertinib, Dasabuvir, MFCD00832476, Leuconicine F inhibit the 3CLPRO. These compounds are stable within the protease proteins' active sites at 20ns MD simulation. The stability is revealed by hydrogen bond formations, hydrophobic interactions, and salt bridge interactions. Our study results also reveal that the selected five compounds against PLPRO and the six compounds against 3CLPRO bind to their active sites with good binding free energy. These compounds that inhibit the activity of PLPRO and 3CLPRO may, therefore, be used for treating COVID-19 infection.
Project description:The spread of a novel coronavirus SARS-CoV-2 and a resulting COVID19 disease in late 2019 has transformed into a worldwide pandemic and has effectively brought the world to a halt. Proteases 3CL<sup>pro</sup> and PL<sup>pro</sup>, responsible for proteolysis of new virions, represent vital inhibition targets for the COVID19 treatment. Herein, we report an <i>in silico</i> docking study of more than 860 COVID19-related compounds from the PubChem database. Molecular dynamic simulations were carried out to validate the conformation stability of compound-ligand complexes with best docking scores. The MM-PBSA approach was employed to calculate binding free energies. The comparison with ca. 50 previously reported potential SARS-CoV-2's proteases inhibitors show a number of new compounds with excellent binding affinities. Anti-inflammatory drugs Montelukast, Ebastine and Solumedrol, the anti-migraine drug Vazegepant or the anti-MRSA pro-drug TAK-599, among many others, all show remarkable affinities to 3CL<sup>pro</sup> and with known side effects present candidates for immediate clinical trials. This study reports thorough docking scores summary of COVID19-related compounds found in the PubChem database and illustrates the asset of computational screening methods in search for possible drug-like candidates. Several yet-untested compounds show affinities on par with reported inhibitors and warrant further attention. Furthermore, the submitted work provides readers with ADME data, ZINC and PubChem IDs, as well as docking scores of all studied compounds for further comparisons.
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:The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - coronavirus disease 2019 (COVID-19) has raised a severe global public health issue and creates a pandemic situation. The present work aims to study the molecular -docking and dynamic of three pertinent medicinal plants <i>i.e. Eurycoma harmandiana</i>, <i>Sophora flavescens</i> and <i>Andrographis paniculata</i> phyto-compounds against SARS-COV-2 papain-like protease (PL<sup>pro</sup>) and main protease (M<sup>pro</sup>)/3-chymotrypsin-like protease (3CL<sup>pro</sup>). The interaction of protein targets and ligands was performed through AutoDock-Vina visualized using PyMOL and BIOVIA-Discovery Studio 2020. Molecular docking with canthin-6-one 9-O-beta-glucopyranoside showed highest binding affinity and less binding energy with both PL<sup>pro</sup> and M<sup>pro</sup>/3CL<sup>pro</sup> proteases and was subjected to molecular dynamic (MD) simulations for a period of 100ns. Stability of the protein-ligand complexes was evaluated by different analyses. The binding free energy calculated using MM-PBSA and the results showed that the molecule must have stable interactions with the protein binding site. ADMET analysis of the compounds suggested that it is having drug-like properties like high gastrointestinal (GI) absorption, no blood-brain barrier permeability and high lipophilicity. The outcome revealed that canthin-6-one 9-O-beta-glucopyranoside can be used as a potential natural drug against COVID-19 protease.
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 sudden increase in the COVID-19 epidemic affected by novel coronavirus 2019 has jeopardized public health worldwide. Hence the necessities of a drug or therapeutic agent that heal SARS-CoV-2 infections are essential requirements. The viral genome encodes a large Polyprotein, further processed by the main protease/ 3C-like protease (3CL<sup>pro</sup>) and papain-like proteases (PL<sup>pro</sup>) into 16 nonstructural proteins to form a viral replication complex. These essential functions of 3CL<sup>pro</sup> and PL<sup>pro</sup> in virus duplication make these proteases a promising target for discovering potential therapeutic candidates and possible treatment for SARS-CoV-2 infection. This study aimed to screen a unique set of protease inhibitors library against 3CL<sup>pro</sup> and PL<sup>pro</sup> of the SARS-CoV-2. A molecular docking study was performed using PyRx to reveal the binding affinity of the selected ligands and molecular dynamic simulations were executed to assess the three-dimensional stability of protein-ligand complexes. The pharmacodynamics parameters of the inhibitors were predicted using admetSAR. The top two ligands (Nafamostat and VR23) based on docking scores were selected for further studies. Selected ligands showed excellent pharmacokinetic properties with proper absorption, bioavailability and minimal toxicity. Due to the emerging and efficiency of remdesivir and dexamethasone in healing COVID-19 patients, ADMET properties of the selected ligands were thus compared with it. MD Simulation studies up to 100 ns revealed the ligands' stability at the target proteins' binding site residues. Therefore, Nafamostat and VR23 may provide potential treatment options against SARS-CoV-2 infections by potentially inhibiting virus duplication though more research is warranted.
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:Coronavirus 3C-like protease (3CL<sup>pro</sup>) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CL<sup>pro</sup> inhibitory activity, but the key constituents with SARS-CoV-2-3CL<sup>pro</sup> 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-2-3CL<sup>pro</sup> inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CL<sup>pro</sup> inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CL<sup>pro</sup> in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CL<sup>pro</sup> 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 3CL<sup>pro</sup>. Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CL<sup>pro</sup>, while the newly identified SARS-CoV-2 3CL<sup>pro</sup> inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.
Project description:3-Chymotrypsin-like protease (3CL<sup>pro</sup>) is a virally encoded main proteinase that is pivotal for the viral replication across a broad spectrum of coronaviruses. This study aims to discover the naturally occurring SARS-CoV-2 3CL<sup>pro</sup> inhibitors from herbal constituents, as well as to investigate the inhibitory mechanism of the newly identified efficacious SARS-CoV-2 3CL<sup>pro</sup> inhibitors. Following screening of the inhibitory potentials of eighty herbal products against SARS-CoV-2 3CL<sup>pro</sup>, Ginkgo biloba leaves extract (GBLE) was found with the most potent SARS-CoV-2 3CL<sup>pro</sup> inhibition activity (IC<sub>50</sub> = 6.68 μg/mL). Inhibition assays demonstrated that the ginkgolic acids (GAs) and the bioflavones isolated from GBLE displayed relatively strong SARS-CoV-2 3CL<sup>pro</sup> inhibition activities (IC<sub>50</sub> < 10 μM). Among all tested constituents, GA C15:0, GA C17:1 and sciadopitysin displayed potent 3CL<sup>pro</sup> inhibition activities, with IC<sub>50</sub> values of less than 2 μM. Further inhibition kinetic studies and docking simulations clearly demonstrated that two GAs and sciadopitysin strongly inhibit SARS-CoV-2 3CL<sup>pro</sup>via a reversible and mixed inhibition manner. Collectively, this study found that both GBLE and the major constituents in this herbal product exhibit strong SARS-CoV-2 3CL<sup>pro</sup> inhibition activities, which offer several promising leading compounds for developing novel anti-COVID-19 medications via targeting on 3CL<sup>pro</sup>.
Project description:SARS-CoV and SARS-CoV-2 encode four structural and accessory proteins (spike, envelope, membrane and nucleocapsid proteins) and two polyproteins (pp1a and pp1ab). The polyproteins are further cleaved by 3C-like cysteine protease (3CL<sup>pro</sup> ) and papain-like protease (PL<sup>pro</sup> ) into 16 nonstructural proteins (nsps). PL<sup>pro</sup> is released from nsp3 through autocleavage, and then it cleaves the sites between nsp1/2, between nsp2/3 and between nsp3/4 with recognition motif of LXGG, and the sites in the C-terminus of ubiquitin and of protein interferon-stimulated gene 15 (ISG15) with recognition motif of RLRGG. Alone or together with SARS unique domain (SUD), PL<sup>pro</sup> can stabilize an E3 ubiquitin ligase, the ring-finger, and CHY zinc-finger domain-containing 1 (RCHY1), through domain interaction, and thus, promote RCHY1 to ubiquitinate its target proteins including p53. However, a dilemma appears in terms of PL<sup>pro</sup> roles. On the one hand, the ubiquitination of p53 is good for SARS-CoV because the ubiquitinated p53 cannot inhibit SARS-CoV replication. On the other hand, the ubiquitination of NF-κB inhibitor (IκBα), TNF receptor-associated factors (TRAFs), and stimulator of interferon gene (STING), and the ISGylation of targeted proteins are bad for SARS-CoV because these ubiquitination and ISGylation initiate the innate immune response and antiviral state. This mini-review analyzes the dilemma and provides a snapshot on how the viral PL<sup>pro</sup> smartly manages its roles to avoid its simultaneously contradictory actions, which could shed lights on possible strategies to deal with SARS-CoV-2 infections.
Project description: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 (3CL<sup>pro</sup>) 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 3CL<sup>pro</sup>. The structure-activity relationships of 9,10-dihydrophenanthrenes as SARS-CoV-2 3CL<sup>pro</sup> 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 3CL<sup>pro</sup> inhibition activity, with IC<sub>50</sub> 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 3CL<sup>pro</sup>via 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 3CL<sup>pro</sup> inhibitor.