Scutellaria baicalensis extract and baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro.
ABSTRACT: 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 (3CLpro) of SARS-CoV-2 is a preferred target for broad spectrum anti-coronavirus drug discovery. We studied the anti-SARS-CoV-2 activity of S. baicalensis and its ingredients. We found that the ethanol extract of S. baicalensis and its major component, baicalein, inhibit SARS-CoV-2 3CLpro activity in vitro with IC50'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 EC50'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 3CLpro activity at µM concentration. All the active compounds and the S. baicalensis extract also inhibit the SARS-CoV 3CLpro, demonstrating their potential as broad-spectrum anti-coronavirus drugs.
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:The 3C-like main protease of SARS-CoV-2 (3CL<sup>Pro</sup>) is responsible for the cleavage of the viral polyprotein. This process is essential for the viral life cycle. Therefore, 3CL<sup>Pro</sup> is a promising target to develop antiviral drugs for COVID-19 prevention and treatment. Traditional enzymatic assays for the identification of 3CL<sup>Pro</sup> inhibitors rely on peptide-based colorimetric or fluorogenic substrates. However, the COVID-19 pandemic has limit or delay access to these substrates, especially for researchers in developing countries attempting to screen natural product libraries. We explored the use of the fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS) as an alternative assay for inhibitor identification. Fluorescence enhancement upon binding of ANS to 3CL<sup>Pro</sup> was observed, and this interaction was competitive with a peptide substrate. The utility of ANS-based competitive binding assay to identify 3CL<sup>Pro</sup> inhibitors was demonstrated with the flavonoid natural products baicalein and rutin. The molecular nature of ANS and rutin interaction with 3CL<sup>Pro</sup> was explored with molecular modeling. Our results suggested that ANS could be employed in a competitive binding assay to facilitate the identification of novel SARS-CoV-2 antiviral compounds.
Project description: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 (3CL<sup>Pro</sup>) 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 3CL<sup>Pro</sup> activity. Methanolic extracts were evaluated in vitro for inhibition of SARS-CoV-2 3CL<sup>Pro</sup> activity using a quenched fluorescence resonance energy transfer (FRET) assay. Extracts from turmeric (<i>Curcuma longa</i>) rhizomes, mustard (<i>Brassica nigra</i>) seeds, and wall rocket <i>(Diplotaxis erucoides</i> subsp. <i>erucoides</i>) at 500 µg mL<sup>-1</sup> displayed significant inhibition of the 3CL<sup>Pro</sup> activity, resulting in residual protease activities of 0.0%, 9.4%, and 14.9%, respectively. Using different extract concentrations, an IC<sub>50</sub> value of 15.74 µg mL<sup>-1</sup> was calculated for turmeric extract. Commercial curcumin inhibited the 3CL<sup>Pro</sup> 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 3CL<sup>Pro</sup> activity. Sinigrin, a major glucosinolate present in mustard seeds and wall rocket, did not have relevant 3CL<sup>Pro</sup> inhibitory activity; however, its hydrolysis product allyl isothiocyanate had an IC<sub>50</sub> value of 41.43 µg mL<sup>-1</sup>. 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.
Project description:The abundance of polyphenols in edible plants makes them an important component of human nutrition. Considering the ongoing COVID-19 pandemic, a number of studies have investigated polyphenols as bioactive constituents. We applied in-silico molecular docking as well as molecular dynamics supported by in-vitro assays to determine the inhibitory potential of various plant polyphenols against an important SARS-CoV-2 therapeutic target, the protease 3CL<sup>pro</sup>. Of the polyphenols in initial in-vitro screening, quercetin, ellagic acid, curcumin, epigallocatechin gallate and resveratrol showed IC<sub>50</sub> values of 11.8 µM to 23.4 µM. In-silico molecular dynamics simulations indicated stable interactions with the 3CL<sup>pro</sup> active site over 100 ns production runs. Moreover, surface plasmon resonance spectroscopy was used to measure the binding of polyphenols to 3CL<sup>pro</sup> in real time. Therefore, we provide evidence for inhibition of SARS-CoV-2 3CL<sup>pro</sup> by natural plant polyphenols, and suggest further research into the development of these novel 3CL<sup>pro</sup> inhibitors or biochemical probes.
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: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.
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), similar to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), which belong to the same Betacoronavirus genus, induces severe acute respiratory disease that is a threat to human health. Since the outbreak of infection by SARS-CoV-2 began, which causes coronavirus disease 2019 (COVID-19), the disease has rapidly spread worldwide. Thus, a search for effective drugs able to inhibit SARS-CoV-2 has become a global pursuit. The 3C-like protease (3CL<sup>pro</sup>), which hydrolyses viral polyproteins to produce functional proteins, is essential for coronavirus replication and is considered an important therapeutic target for diseases caused by coronaviruses, including COVID-19. Many 3CL<sup>pro</sup> inhibitors have been proposed and some new drug candidates have achieved success in preclinical studies. In this review, we briefly describe recent developments in determining the structure of 3CL<sup>pro</sup> and its function in coronavirus replication and summarise new insights into 3CL<sup>pro</sup> inhibitors and their mechanisms of action. The clinical application prospects and limitations of 3CL<sup>pro</sup> inhibitors for COVID-19 treatment are also discussed.
Project description: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, PL<sup>PRO</sup> and 3CL<sup>PRO</sup>, 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 PL<sup>PRO</sup> and 3CL<sup>PRO</sup> proteases. After thoroughly analyzing the screening results, we found five compounds, Bemcentinib, Pacritinib, Ergotamine, MFCD00832476, and MFCD02180753 inhibit PL<sup>PRO</sup> and six compounds, Bemcentinib, Clofazimine, Abivertinib, Dasabuvir, MFCD00832476, Leuconicine F inhibit the 3CL<sup>PRO</sup>. 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 PL<sup>PRO</sup> and the six compounds against 3CL<sup>PRO</sup> bind to their active sites with good binding free energy. These compounds that inhibit the activity of PL<sup>PRO</sup> and 3CL<sup>PRO</sup> may, therefore, be used for treating COVID-19 infection.
Project description:The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emphasized the urgency to develop effective therapeutics. Drug repurposing screening is regarded as one of the most practical and rapid approaches for the discovery of such therapeutics. The 3C like protease (3CL pro ), or main protease (M pro ) of SARS-CoV-2 is a valid drug target as it is a specific viral enzyme and plays an essential role in viral replication. We performed a quantitative high throughput screening (qHTS) of 10,755 compounds consisting of approved and investigational drugs, and bioactive compounds using a SARS-CoV-2 3CL pro assay. Twenty-three small molecule inhibitors of SARS-CoV-2 3CL pro have been identified with IC50s ranging from 0.26 to 28.85 ?M. Walrycin B (IC 50 = 0.26 µM), Hydroxocobalamin (IC 50 = 3.29 µM), Suramin sodium (IC 50 = 6.5 µM), Z-DEVD-FMK (IC 50 = 6.81 µM), LLL-12 (IC 50 = 9.84 µM), and Z-FA-FMK (IC 50 = 11.39 µM) are the most potent 3CL pro inhibitors. The activities of anti-SARS-CoV-2 viral infection was confirmed in 7 of 23 compounds using a SARS-CoV-2 cytopathic effect assay. The results demonstrated a set of SARS-CoV-2 3CL pro inhibitors that may have potential for further clinical evaluation as part of drug combination therapies to treating COVID-19 patients, and as starting points for chemistry optimization for new drug development.