Discovery of naturally occurring inhibitors against SARS-CoV-2 3CLpro from Ginkgo biloba leaves via large-scale screening.
ABSTRACT: 3-Chymotrypsin-like protease (3CLpro) 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 3CLpro inhibitors from herbal constituents, as well as to investigate the inhibitory mechanism of the newly identified efficacious SARS-CoV-2 3CLpro inhibitors. Following screening of the inhibitory potentials of eighty herbal products against SARS-CoV-2 3CLpro, Ginkgo biloba leaves extract (GBLE) was found with the most potent SARS-CoV-2 3CLpro inhibition activity (IC50 = 6.68 μg/mL). Inhibition assays demonstrated that the ginkgolic acids (GAs) and the bioflavones isolated from GBLE displayed relatively strong SARS-CoV-2 3CLpro inhibition activities (IC50 < 10 μM). Among all tested constituents, GA C15:0, GA C17:1 and sciadopitysin displayed potent 3CLpro inhibition activities, with IC50 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 3CLprovia 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 3CLpro inhibition activities, which offer several promising leading compounds for developing novel anti-COVID-19 medications via targeting on 3CLpro.
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 recent appearance of COVID-19 virus has created a global crisis due to unavailability of any vaccine or drug that can effectively and deterministically work against it. Naturally, different possibilities (including herbal medicines having known therapeutic significance) have been explored by the scientists. The systematic scientific study (beginning with in silico study) of herbal medicines in particular and any drug in general is now possible as the structural components (proteins) of COVID-19 are already characterized. The main protease of COVID-19 virus is M<sup>pro</sup> or 3CL<sup>pro</sup> which is a key CoV enzyme and an attractive drug target as it plays a pivotal role in mediating viral replication and transcription. In the present study, 3CL<sup>pro</sup> is used to study drug:3CL<sup>pro</sup> interactions and thus to investigate whether all or any of the main chemical constituents of <i>Tinospora cordifolia</i> (e.g. berberine (C<sub>20</sub>H<sub>18</sub>NO<sub>4</sub>), β-sitosterol (C<sub>29</sub>H<sub>50</sub>O), coline (C<sub>5</sub>H<sub>14</sub>NO), tetrahydropalmatine (C<sub>21</sub>H<sub>25</sub>NO<sub>4</sub>) and octacosanol (C<sub>28</sub>H<sub>58</sub>O)) can be used as an anti-viral drug against SARS-CoV-2. The in silico study performed using tools of network pharmacology, molecular docking including molecular dynamics have revealed that among all considered phytochemicals in <i>Tinospora cordifolia</i>, berberine can regulate 3CL<sup>pro</sup> protein's function due to its easy inhibition and thus can control viral replication. The selection of <i>Tinospora cordifolia</i> was motivated by the fact that the main constituents of it are known to be responsible for various antiviral activities and the treatment of jaundice, rheumatism, diabetes, etc. Communicated by Ramaswamy H. Sarma.
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 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: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: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:After the emergence of the pandemic, repurposed drugs have been considered as a quicker way of finding potential antiviral agents. SARS-CoV-2 3CL<sup>pro</sup> is essential for processing the viral polyproteins into mature non-structural proteins, making it an attractive target for developing antiviral agents. Here we show that Vitamin K3 screened from the FDA-Approved Drug Library containing an array of 1,018 compounds has potent inhibitory activity against SARS-CoV-2 3CL<sup>pro</sup> with the IC<sub>50</sub> value of 4.78 ± 1.03 μM, rather than Vitamin K1, K2 and K4. Next, the time-dependent inhibitory experiment was carried out to confirm that Vitamin K3 could form the covalent bond with SARS-CoV-2 3CL<sup>pro</sup>. Then we analyzed the structure-activity relationship of Vitamin K3 analogues and identified 5,8-dihydroxy-1,4-naphthoquinone with 9.8 times higher inhibitory activity than Vitamin K3. Further mass spectrometric analysis and molecular docking study verified the covalent binding between Vitamin K3 or 5,8-dihydroxy-1,4-naphthoquinone and SARS-CoV-2 3CL<sup>pro</sup>. Thus, our findings provide valuable information for further optimization and design of novel inhibitors based on Vitamin K3 and its analogues, which may have the potential to fight against SARS-CoV-2.
Project description:The main protease, M<sup>pro</sup> (or 3CL<sup>pro</sup>) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide. Feline infectious peritonitis, a fatal coronavirus infection in cats, was successfully treated previously with a prodrug GC376, a dipeptide-based protease inhibitor. Here, we show the prodrug and its parent GC373, are effective inhibitors of the M<sup>pro</sup> from both SARS-CoV and SARS-CoV-2 with IC<sub>50</sub> values in the nanomolar range. Crystal structures of SARS-CoV-2 M<sup>pro</sup> with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 replication in cell culture. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals. The work here lays the framework for their use in human trials for the treatment of COVID-19.
Project description:The COVID-19 disease caused by the SARS-CoV-2 has emerged as a worldwide pandemic and caused huge damage to the lives and economy of more than hundred countries. As on May 10, 2020, more than 4,153,300 people stand infected from the virus due to an unprecedented rate of transmission and 282,700 have lost their lives because of the disease. In this context, medicinal plants may provide a way to treat the disease by targeting specific essential proteins of the virus. We screened about 51 medicinal plants and found that Tea (Camellia sinensis) and Haritaki (Terminalia chebula) has potential against SARS-COV-2 3CL<sup>pro</sup> , with an IC<sub>50</sub> for Green Tea as 8.9 ± 0.5 μg/ml and Haritaki 8.8 ± 0.5 μg/ml. The in-silico studies suggested that Tea component Thearubigins binds to the cysteine 145 of protease active site and could be a pharmacoactive molecule. We predict that the inhibition in protease activity may be able to halt the SARS-CoV-2 replication cycle and therefore, we propose Green Tea, Black Tea, and Haritaki plant extracts as potential therapeutic candidates for SARS-CoV-2 infection. Further investigation on role of bioactive constituents of extracts is needed to establish the molecular basis of inhibition and towards expedited drug discovery.
Project description:Since the first report of a new pneumonia disease in December 2019 (Wuhan, China) the WHO reported more than 148 million confirmed cases and 3.1 million losses globally up to now. The causative agent of COVID-19 (SARS-CoV-2) has spread worldwide, resulting in a pandemic of unprecedented magnitude. To date, several clinically safe and efficient vaccines (e.g., Pfizer-BioNTech, Moderna, Johnson & Johnson, and AstraZeneca COVID-19 vaccines) as well as drugs for emergency use have been approved. However, increasing numbers of SARS-Cov-2 variants make it imminent to identify an alternative way to treat SARS-CoV-2 infections. A well-known strategy to identify molecules with inhibitory potential against SARS-CoV-2 proteins is repurposing clinically developed drugs, e.g., antiparasitic drugs. The results described in this study demonstrated the inhibitory potential of quinacrine and suramin against SARS-CoV-2 main protease (3CL<sup>pro</sup>). Quinacrine and suramin molecules presented a competitive and noncompetitive inhibition mode, respectively, with IC<sub>50</sub> values in the low micromolar range. Surface plasmon resonance (SPR) experiments demonstrated that quinacrine and suramin alone possessed a moderate or weak affinity with SARS-CoV-2 3CL<sup>pro</sup> but suramin binding increased quinacrine interaction by around a factor of eight. Using docking and molecular dynamics simulations, we identified a possible binding mode and the amino acids involved in these interactions. Our results suggested that suramin, in combination with quinacrine, showed promising synergistic efficacy to inhibit SARS-CoV-2 3CL<sup>pro</sup>. We suppose that the identification of effective, synergistic drug combinations could lead to the design of better treatments for the COVID-19 disease and repurposable drug candidates offer fast therapeutic breakthroughs, mainly in a pandemic moment.