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Discovery of naturally occurring inhibitors against SARS-CoV-2 3CLpro from Ginkgo biloba leaves via large-scale screening

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.


Introduction
The pandemic of coronavirus disease 2019 (COVID- 19), an unprecedented disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is sweeping expeditiously all over the world [1,2]. Owing to the high infectivity and lack of efficacious drugs, COVID-19 is putting unprecedented pressures to public health, economic development, and global safety [3]. As to April 12th, 2021, COVID-19 has infected over 136.5 million people and has killed more than 2.9 million people worldwide [4]. Currently, scientists around the world are trying to find efficacious therapeutics for combating COVID-19, via targeting on several validated therapeutic targets [5]. Among all identified therapeutic targets for treating COVID-19, the conserved 3-chymotrypsinlike protease (3CL pro ), a key enzyme responsible for coronavirus replication, has been validated as a pivotal therapeutic target for fighting this pandemic [6]. 3CL pro is a virally encoded main proteinase that implicated in the maturation of the functional proteases by cleaving the viral polyproteins [7,8], dysfunction or strong inhibition on 3CL pro will block the replication of multiple coronaviruses.
The crucial role of 3CL pro in coronavirus replication has aroused great interests in discovery of efficacious 3CL pro inhibitors as the leading compounds for the development of novel antiviral agents. Although a wide range of phytochemicals and synthetic molecules have been reported with anti-3CL pro activity [9,10], the efficacious SARS-CoV-2 3CL pro inhibitors with high potency and favorable safety profiles are rarely reported. Thus, it is an urgent need for discovering more efficacious SARS-CoV-2 3CL pro inhibitors to develop novel antiviral agents to fight COVID-19. It is well-known that the phytochemicals from medicinal plants remain one of the major sources for discovery of the leading compounds for drug development [11], while some natural compounds (such as flavonoids [12], triterpenes [13], and phenolic compounds [14]) have been reported with 3CL pro inhibition activity, encouraging us to find more potent SARS-CoV-2 3CL pro inhibitors from herbal medicines. For this purpose, a large-scale screening campaign was performed by us, in which a total of 80 herbal products were collected and the inhibition potentials of these herbal products against SARS-CoV-2 3CL pro were assayed via a biochemical approach. After screening, we noticed that the Ginkgo biloba leaves extract (GBLE) displayed the most potent SARS-CoV-2 3CL pro inhibition activity (IC 50 = 6.68 μg/mL), which encouraged us to further characterize the key constituents in Ginkgo biloba leaves responsible for SARS-CoV-2 3CL pro inhibition.
This study intended to assay the inhibition potentials for the major phytochemicals isolated from GBLE, as well as to explore the inhibitory mechanisms of the newly identified SARS-CoV-2 3CL pro inhibitors from this herbal product. For these purposes, a total of twenty natural products isolated from Ginkgo biloba leaves were collected, while the inhibitory potentials of these compounds against SARS-CoV-2 3CL pro were carefully assayed. Among all tested natural compounds, the bioflavones and ginkgolic acids (GAs) were identified as strong SARS-CoV-2 3CL pro inhibitors (IC 50 < 10 μM). To further reveal the inhibitory mechanisms of the newly identified anti-SARS-CoV-2 3CL pro molecules, inhibition kinetic analyses and docking simulations were performed. All these studies are helpful for deciphering the key active ingredients in GBLE responsible for SARS-CoV-2 3CL pro inhibition, which will facilitate the development of novel anti-COVID-19 agents deriving from natural products.

Expression and purification of SARS-CoV-2 3CL pro
A Smt3-SARS-CoV-2 3CL pro fusion construct was Escherichia coli (E. coli) codon optimized and cloned into the pET29a (+) vector [17,18]. The 3CL pro cleavage site (SAVLQS↓GFRK) located at the N-terminus of SARS-CoV-2 3CL pro . The C-terminus of SARS-CoV-2 3CL pro was followed by a modified HRV 3C protease cleavage site (SGVTFQ↓GP) and a His6tag. The expression of SARS-CoV-2 3CL pro was induced in autoinduction method in E. coli stain BL21 (DE3) at 18 • C. After 36 h of induction, cells were harvested by centrifugation. Cell pellet was resuspended in lysis buffer (pH 8.0, 25 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, 0.1 mg/mL lysozyme, 25 U/mL SuperNuclease) and incubated for 30 min at room temperature. Cells were lysed by sonication on ice, then cell debris was removed by centrifugation at 18000 rpm at 4 • C for 30 min. The supernatant was collected and purified by Ni-NTA agarose. The SARS-CoV-2 3CL pro was purified in further on a Superdex 200 10/300 GL column in protein storage buffer (pH 7.4, 25 mM HEPES, 150 mM NaCl, 1 mM DTT).
The hydrolytic product emits strong fluorescence signals around 490 nm, which can be recorded by a microplate reader (SpectraMax® iD3, Molecular Devices, Austria).

Determination of inhibition constants of GA C15:0, GA C17:1, and sciadopitysin
The inhibition kinetics were performed a set of inhibition assay, in which increasing concentrations of each inhibitor (GA C15:0, GA C17:1 or sciadopitysin) and various concentrations of Dabcyl-KNSTLQSGLRKE-Edans were used. The data were analyzed according to the equations of various inhibition kinetic modes (competitive mode, non-competitive mode, and mixed mode), while the second plot of Lineweaver-Burk plots slopes were used to determine the inhibition constant (K i ) values, as previously reported [20,21].

Molecular docking simulations
The structure of SARS-CoV-2 3CL pro was downloaded from the Protein Data Bank (PDB Code: 6XHU) [22]. AutoDockTools 1.5.6 was used for the preparation of input files of SARS-CoV-2 3CL pro and the ligands by adding and merging non-polar hydrogen atoms and adjusting charges. Druggable cavities of SARS-CoV-2 3CL pro were recommended by CavityPlus [23]. The implementation of docking simulations was using AutoDock Vina (1.1.2). The grid boxes were placed at the catalytic binding pockets and recommended cavities. The output files were imported into Discovery Studio Visualizer 2020 software in order to check ligand-receptor interactions.

Statistical analysis
The IC 50 values and the K i values were evaluated by nonlinear regression using GraphPad Prism 7.0 software (GraphPad Software, Inc., La Jolla, USA).

Screening of the inhibition potentials of 80 herbs against SARS-CoV-2 3CL pro
Firstly, the inhibition potentials of 80 herbal products (100 μg/mL, final concentration) on SARS-CoV-2 3CL pro were screened by using Dabcyl-KNSTLQSGLRKE-Edans as a FRET-based substrate. As depicted in Fig. 1, among all tested herbal products, GBLE displayed the most potent 3CL pro inhibition activity, and this herbal product could inhibit nearly 70 % hydrolytic activity of SARS-CoV-2 3CL pro at 100 μg/mL. To further quantify the inhibitory activity of GBLE, the dose-inhibition curve of GBLE on SARS-CoV-2 3CL pro was plotted using increasing dosages (from 1.25 to 100 μg/mL). As shown in Fig. 2, GBLE dosedependently inhibited the target enzyme, with the calculated IC 50 value of 6.68 μg/mL. This finding suggests that GBLE contains naturally occurring inhibitors against SARS-CoV-2 3CL pro .

Inhibition kinetics of three newly identified SARS-CoV-2 3CL pro inhibitors
To further investigate the inhibitory mechanisms of the newly identified 3CL pro inhibitors in GBLE, three potent SARS-CoV-2 3CL pro inhibitors (GA C15:0, GA C17:1 and sciadopitysin) isolated from GBLE were selected for inhibition kinetic studies. As shown in Fig. 5, both inhibition kinetic plots and Lineweaver-Burk plots of these three agents against SARS-CoV-2 3CL pro clearly demonstrated that two GAs (C15:0 & C17:1) and sciadopitysin strongly inhibited SARS-CoV-2 3CL pro via a mixed-inhibition manner, with K i values of 0.73 μM, 1.02 μM and 2.96 μM for GA C15:0, GA C17:1, and sciadopitysin, respectively. These results suggest that the GAs and sciadopitysin act as mixed-type inhibitors against SARS-CoV-2 3CL pro , implying that these agents may bind on this key target enzyme at both catalytic site and another non-catalytic site.

Molecular docking simulations
To gain deeper insights into the inhibitory mechanisms of the GAs and sciadopitysin against SARS-CoV-2 3CL pro , a dimer crystal structure of SARS-CoV-2 3CL pro was used for docking simulations [22]. Firstly, the possible ligand-binding pockets in SARS-CoV-2 3CL pro were assessed by utilizing the ligandability and druggability scores calculated by Cav-ityPlus. As shown in Fig. 6 and Table S1, three druggable pockets (the V site located at the dimer interface, and two catalytic sites of both chain A and chain B) were identified as possible ligand-binding pockets of this key enzyme. Docking simulations demonstrated that GA C15:0, GA C17:1 and sciadopitysin could be well-docked into both the V site and the catalytic sites of SARS-CoV-2 3CL pro (Fig. 7), while the predicted binding energies of three compounds binding on either the V site or the catalytic site were listed in Table S2.
The key interactions between three newly identified 3CL pro inhibitors and SARS-CoV-2 3CL pro were also analyzed. As shown in Fig. S7 and Fig. S8, the GAs (including C15:0 and C17:1) created strong interactions with some residuals surrounding on the V site mainly on hydrophobic interactions and hydrogen bonding. Meanwhile, these two hydrophobic compounds could also occupy the catalytic site of SARS-CoV-2 3CL pro via hydrophobic interactions and hydrogen bond interactions, as well as the Pi-sulfur interaction with the key catalytic residue (Cys145). By contrast, sciadopitysin interacted with the residuals surrounding on both the V site and the catalytic site mainly on hydrophobic interactions and hydrogen bonding (Fig. S9). Notably, the phenolic groups of sciadopitysin at both the C-19 and the C-21 sites played crucial roles in binding on SARS-CoV-2 3CL pro , owing to that the C-19 and the C-21 phenolic groups could create strong interactions with Asp216 and Asn119 for the catalytic site and the V site, respectively.
Currently, there are limited therapeutic options available to treat COVID-19 in clinical settings. To fight against COVID-19 effectively, the medicinal chemists have made great endeavors to discover efficacious agents for blocking replication of COVID-19 virus via targeting on several validated therapeutic targets [6]. Among all identified therapeutic targets for treating COVID-19, 3CL pro is a promising therapeutic target owing to its indispensable role in the replication of this new coronavirus [25]. Until now, the efficacious 3CL pro inhibitors with high potency and favorable safety profiles are rarely reported. Thus, there is an urgent need for discovering more efficacious 3CL pro inhibitors for the development of therapeutic agents for combating COVID-19. To find naturally occurring 3CL pro inhibitors, a screening campaign was performed to discover the herbal medicine(s) with anti-SARS-CoV-2 3CL pro activity. Within all tested eighty herbal products, GBLE exhibited the most potent SARS-CoV-2 3CL pro inhibition activity, with the IC 50 value of 6.68 μg/mL. This finding encouraged us to identify the key bioactive constituents in GBLE responsible for SARS-CoV-2 3CL pro inhibition, as well as to investigate the inhibitory mechanisms of these newly identified naturally occurring 3CL pro inhibitors.
As a popular herb used in both western and eastern countries, Ginkgo biloba leaves have been widely applied for preventing and treating a variety of human diseases, including cardiovascular disorders, pulmonary disease, and central nervous system diseases [26][27][28]. Increasing evidence has indicated that some major constituents (such as bioflavones and GAs) in GBLE exhibited broad antiviral activities against a wide range of DNA (such as human Cytomegalovirus) and RNA (such as SARS-CoV, Ebola virus and human immunodeficiency virus) viruses [12,[29][30][31]. However, the anti-SARS-CoV-2 effects of GBLE have not been reported yet. In this study, our findings revealed that both GBLE and several phytochemicals in this herbal extract (such as the bioflavones and GAs) could strongly inhibit the hydrolytic activity of 3CL pro , suggesting that GBLE and its major phytochemicals might block the replication of SARS-CoV-2 via targeting this pivotal enzyme. In addition to the anti-3CL pro activity, previous studies have revealed that GBLE constituents (such as the bioflavones) could modulate the blood coagulation and ameliorates inflammation both in vitro and in vivo [28,[32][33][34]. It is well-known that COVID-19 is a complex, multi-organ and heterogeneous illness, the severe disease cases are frequently accompanied by the hypercoagulable inflammatory state [35,36]. As one of the most popular herbal medicines, Ginkgo biloba leave extract possess a variety of beneficial effects, such as anti-coagulate, anti-inflammatory, anti-oxidative lowing blood pressure, anti-obesity, and other biological activities [26,32,[37][38][39]. Thus, it is easily conceivable  that GBLE may bring significant benefits to the COVID-19 patients with certain underlying medical conditions (such as cardiovascular disease, stroke, high blood pressure, diabetes, and obesity), by alleviating the major symptoms of both COVID-19 and other underlying health problems.
It is well-known that the bioflavones are the major class of bioactive constitutes in GBLE. Previous studies have reported that the bioflavones isolated from GBLE and other medicinal plants exhibited a wide range of beneficial effects, including anti-thrombin, anti-oxidative, antiinflammatory, and antiviral activities [28,31,32,40]. It also has been    reported that the bioflavones displayed strong SARS-CoV 3CL pro inhibition potency [31]. This study found that the bioflavones isolated from GBLE displayed strong inhibition against SARS-CoV-2 3CL pro potency. These findings suggested that the natural bioflavones could act as promising leading compounds for developing anti-COVID-19 or the broad-spectrum anti-CoVs agents. Nevertheless, most naturally occurring bioflavones are found with poor membrane permeability, poor solubility, and extremely poor oral bioavailability [32]. To develop more efficacious orally administrated 3CL pro inhibitors, the natural bioflavones should be extensively modified to simultaneously improve the inhibition potency and the drug-likeness properties. Another strategy is to develop the nasal administration systems for delivery of the bioactive bioflavones to lung, for blocking replication of COVID-19 virus at this target organ and then alleviating the major symptoms of COVID-19. In addition to bioflavones, GAs in GBLE were also found with strong SARS-CoV-2 3CL pro inhibition activities (IC 50 < 5 μM). Accumulating evidence has demonstrated that GAs possess a wide range of antiviral effects via disturbing the viral replication [29,30,41]. Generally, the levels of toxic ginkgolic acids in marketed Ginkgo biloba products are strictly limited lowering than 5 ppm, owing to that these agents could cause severe allergic reactions [42]. In these cases, GAs is not recommended as the orally administrated agents for treating COVID-19, but these abandoned ingredients could be used as the disinfection or cleaning products for external use. Structurally, the GAs isolated from GBLE bear a hydrophobic long chain and these compounds could be easily modified as the surfactants. In the future, new antiviral surfactants could be synthesized and developed using GAs as the starting materials, which also can be used in combination with other marketed disinfectants for the prevention and disinfection of broad spectrum of coronaviruses in vitro.

Conclusion
In summary, this study revealed the key ingredients in Ginkgo biloba leaves extract (GBLE) responsible for SARS-CoV-2 3CL pro inhibition and investigated the inhibitory effects of the newly identified SARS-CoV-2 3CL pro inhibitors isolated from GBLE. Following assessing the inhibitory potentials of twenty phytochemicals isolated from GBLE against SARS-CoV-2 3CL pro , the bioflavones and the GAs were found with efficacious SARS-CoV-2 3CL pro inhibition activities (IC 50 < 10 μM). Among all tested phytochemicals in GBLE, two GAs (GA C15:0, GA C17:1) and a bioflavone (sciadopitysin) displayed the most potent SARS-CoV-2 3CL pro inhibition activities. Inhibition kinetic analyses demonstrated these three newly identified 3CL pro inhibitors strongly inhibit SARS-CoV-2 3CL pro in a reversible and mixed-inhibition manner, with K i values of 0.73 μM, 1.02 μM and 2.96 μM, for GA C15:0, GA C17:1, and sciadopitysin respectively. Docking simulations showed that two GAs and sciadopitysin could be well-docked into two druggable pockets (the V site and the catalytic sites) of SARS-CoV-2 3CL pro , which agreed well with the mixed-inhibition modes of these three agents. Collectively, our findings revealed the key constituents in GBLE responsible for SARS-CoV-2 3CL pro inhibition, which offered several promising lead compounds for developing novel 3CL pro inhibitors to treat COVID-19 or other coronavirus.