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In Silico Screening of Potential Spike Glycoprotein Inhibitors of SARS-CoV-2 with Drug Repurposing Strategy

Objective To select potential molecules that can target viral spike proteins, which may potentially interrupt the interaction between the human angiotension-converting enzyme 2 (ACE2) receptor and viral spike protein by virtual screening. Methods The three-dimensional (3D)-coordinate file of the receptor-binding domain (RBD)-ACE2 complex for searching a suitable docking pocket was firstly downloaded and prepared. Secondly, approximately 15,000 molecular candidates were prepared, including US Food and Drug Administration (FDA)-approved drugs from DrugBank and natural compounds from Traditional Chinese Medicine Systems Pharmacology (TCMSP), for the docking process. Then, virtual screening was performed and the binding energy in Autodock Vina was calculated. Finally, the top 20 molecules with high binding energy and their Chinese medicine (CM) herb sources were listed in this paper. Results It was found that digitoxin, a cardiac glycoside in DrugBank and bisindigotin in TCMSP had the highest docking scores. Interestingly, two of the CM herbs containing the natural compounds that had relatively high binding scores, Forsythiae fructus and Isatidis radix, are components of Lianhua Qingwen (莲花清瘟), a CM formula reportedly exerting activity against severe acute respiratory syndrome (SARS)-Cov-2. Moreover, raltegravir, an HIV integrase inhibitor, was found to have a relatively high binding score. Conclusions A class of compounds, which are from FDA-approved drugs and CM natural compounds, that had high binding energy with RBD of the viral spike protein. Our work provides potential candidates for other researchers to identify inhibitors to prevent SARS-CoV-2 infection, and highlights the importance of CM and integrative application of CM and Western medicine on treating COVID-19.

To date, COVID-19 cases have been reported in more than 200 countries. As of July 20, 2020, a cumulative total of 14,340,653 COVID-19 cases were reported worldwide with 603,111 death. Although viral nucleic acid detection kits have been rapidly developed, effective drugs and vaccines are still being studied and are urgently needed. Facing such urgent situations, although necessary, developing new drugs or vaccines will require a very long time to complete, and drug repositioning is more rapid and possible to discover anti-SARS-CoV-2 drugs from US Food and Drug Administration (FDA)-approved drugs. (3) Chinese medicine (CM) has been applied in combating virus-related diseases such as SARS and H1N1 (4) and a large number of studies indicated the therapeutic effects of integrative application of Western medicine (WM) and CM were significantly better than the simple application of WM. (5)(6)(7)(8)(9)(10) Essentially, although the therapeutic components are still unclear, Lianhua Qingwen (连花清瘟), (11) Toujie Quwen Granule (透解祛 瘟颗粒), and Qingfei Paidu Decoction (清肺排毒汤) are playing an important role in the treatment of COVID-19. (12,13) Thus, rapid screening from these sources is a promising approach for discovering anti-COVID-19 drugs. (4,14) Receptor recognition by coronavirus is the fi rst and essential step for entering human cells. The homotrimeric spike glycoprotein (S protein), located on the envelope of SARS-CoV-2, is responsible for receptor recognition. The S1 subunit of the S protein, containing the receptor-binding domain (RBD), directly interacts with the receptor on the human cell membrane, while the S2 subunit facilitates virus-cell fusion and entry. (15) Angiotensin-converting enzyme 2 (ACE2) was shown to be the receptor mediating SARS-CoV-2 invasion in human cells. (1) Thus, interrupting the interaction between the S protein and ACE2 is a strategy to inhibit virus entry. (16) Recently, three-dimensional (3D) structures of the homotrimeric spike glycoprotein (17) and the ACE2-B0AT1 complex (18) were solved by cryo-EM respectively. In addition, the complex structures of the receptor-binding domain (RBD) of the S protein bound to the human ACE2 were independently reported by several research teams. (19)(20)(21) These accurate coordinates of protein structures make it possible for avoiding inaccurate protein structure modeling and accelerating drug screening and vaccine design. Pharmacology (TCMSP) databases to identify potential inhibitors targeting the RBD of viral spike proteins. The selected binding pocket neighbors the interface between the viral spike protein and human receptor ACE2, which may further inhibit their interaction to prevent the virus from invading human cells. Following this computational work, more vigorous in vitro and in vivo experiments need to be performed. We hope this work will contribute to the discovery of anti-COVID-19 drugs.

METHODS
DrugBank is a drug data resource that contains comprehensive drug information, including approved and experimental small-molecule, biologics, and nutraceutical data. (22) The latest version of DrugBank was released on January 13, 2020. It currently has 2,628 approved small drugs. Before performing virtual screening, approved drugs with molecular weights larger than 500 kDa, including polypeptides, were deleted; thus, only 2,191 FDA-approved drugs were screened. TCMSP was developed by Northwest University in China, which is a platform providing information connecting bioactive molecules in CM, targets, and diseases in a network. In addition, this platform also provides related pharmacokinetic properties. (23) Based on this platform, 13,026 small molecules from CM herbs were screened. All molecular structure fi les were optimized by the force fi eld MMFF94.
AutoDock Vina 1.1.2 (24) (25) and Discovery Studio 2016 (26)  water molecules with solvent layers 10 Å between the box edges and solute surface. The AMBER FF14SB force fi elds were applied and the SHAKE algorithm was used to restrict all covalent bonds involving hydrogen atoms with a time step of 2 fs. The particle mesh Ewald method (28) was employed to treat long-range electrostatic interactions. For each solvated system, two steps of minimization were performed before the heating step. The first 4,000 cycles of minimization were performed with all heavy atoms restrained with the force constant 50 kcal/(mol˙Å 2 ), whereas solvent molecules and sodium/chlorine counter ions were free to move. Then, non-restrained minimization was carried out involving 2,000 cycles of steepest descent minimization followed by 2,000 cycles of conjugated gradient minimization. Afterwards, the whole system was fi rst heated from 0 to 300 K in 50 ps using Langevin dynamics at a constant volume and, then, equilibrated for 400 ps at a constant pressure of 1 atm. A weak constraint of 10 kcal/(mol˙Å 2 ) was used to restrain all the heavy atoms during the heating steps. Periodic boundary dynamics simulations were carried out for the whole system with an NPT (constant composition, pressure and temperature) ensemble at a constant pressure of 1 atm and 300 K in the production step. In production phase, 100 ns simulation was carried out. The binding free energy of complex was calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. (29)

Binding Pocket Identified on RBD of Spike Protein
The second-ranked pocket (called Site 2) was selected as the drug target for further ligand docking because it is near the protein-protein interface and may thus induce a conformational change to intervene in the interaction ( Figure 1). Site 2 actually has the highest score among the 3 sites. The coordination of the pocket is as follows: center x=-56.387, center y=52.408, center z=21.937; size x=25, size y=25, size z=25.

Docking Results of Molecules from DrugBank Dataset
For the DrugBank dataset, Table 1  found that digitoxin is also a potential anticancer drug. (30,31) However, digitoxin can cause toxicity to the human body, including causing nausea, anorexia, confusion and so on.

Docking Results of TCMSP Dataset
The top 10 binding results of the TCMSP dataset are shown in Table 2. The complete binding scores information of TCMSP are available upon request.  Lsatis indigotica is a folk medicine used to treat viral disease and inflammatory disease. (32) In addition, Wei, et al (33) reported that bisindigotin can act as an antagonist to relieve the hepatoxicity caused by 2,3,7,8-tetrachlorodibenzo-pdioxin, which is a carcinogen.
The result of RMSD also refl ect that the docking between digitoxin and S protein is stable (Figure 3). In addition, 100-ns MD simulation was also performed to verify the reliability of binding between bisindigotin and viral S protein. However, the result was not convergent.

DISCUSSION
The world is now confronted with a public health crisis caused by SARS-CoV-2. Normally, de novo drug development is a time-consuming project. Therefore, drug repurposing may be helpful to find the drug observed that the cavity neighboring the interface between the viral spike protein and the human ACE2 receptor is the second-ranked pocket. Targeting this position may contribute to interrupting the interaction between the S protein and the ACE2 receptor. Due to its high ranking and position advantages, we used this cavity for the next  Here, we firstly chose the spike protein rather than the ACE2 receptor for a binding pocket search because ACE2 is expressed in various types of human cells, as a result, targeting ACE2 might cause more side effects. We   Additionally, in the top-10 list of DrugBank docking approach to treat COVID-19 disease. In the future research, in vitro tests will be carried out to verify the binding between the S protein and the molecules identifi ed by the computational methods as well as their inhibitory abilities against the interaction between RBD and ACE2 receptors.

Confl icts of Interest
The authors declare no confl ict of interest.