Screening of novel antiviral compounds from the natural product library of traditional Chinese medicine based on the structure of Ebola virus glycoprotein monomers
Ebola virus (EBOV) is a enveloped filamentous virus that causes acute hemorrhagic fever, which is a zoonotic infectious disease commonly found in primates, with a mortality rate of up to 50% to 90%. In recent years, there have been several severe Ebola outbreaks in West Africa, but so far no specific anti Ebola hemorrhagic fever treatment drugs have been approved for sale. The entry of EBOV into host cells is a critical step in infection, mediated by the viral surface transmembrane single glycoprotein of EBOV sgP. This type I fusion protein contains two subunits connected by disulfide bonds, namely GP1 exposed on the surface and GP2 embedded in the viral membrane. After binding to the host cell through cell surface attachment factors, the virus is internalized, and the corresponding cysteine proteases and proteases in the endosome cleave most of the peptide chains of the GP1 subunit, exposing the receptor binding site. This conformational transition of GP facilitates host virus membrane fusion and subsequent release of the virus genome. This fusion mechanism is similar to HIV-1 virus and influenza virus. Therefore, developing inhibitor molecules that effectively interfere with the conformational transition of EBOV sgP can help block virus invasion and replication.

In 2019, Elizabeth E. Fry and her team from the Department of Structural Biology at the University of Oxford in the UK successfully prepared EBOV sgP protein using protein expression purification crystallization technology, and determined its three-dimensional structure using X-ray diffraction experiments (RCSB Protein Data Bank ID: 5JQ7), providing a computer virtual screening model. In addition, due to the unique structure and diversity of natural products, especially traditional Chinese medicine ingredient libraries, they have been used to treat various chronic and infectious diseases, and can also be used to explore anti Ebola virus inhibitors. Based on the above considerations, this study adopts computer simulation technology to screen EBOV sgP inhibitor molecules from the natural product library of traditional Chinese medicine.

Ahmad’s research group first targeted the three-dimensional structure of EBOV-GP transmembrane glycoprotein trimer cavities based on the Mcule compound library in 2017（ https://mcule.com/database /Conduct large-scale virtual screening of inhibitors using two molecular docking software, Auto Dock Vina and Flex-X, combined with online analysis tool admetSAR（ http://www.admetexp.org ）ADMET filtering was performed and three hit molecules were found. Since 2000, there have been continuous outbreaks of Ebola virus, and the research and development of antiviral molecules remains a global pharmaceutical research hotspot. Therefore, in order to mine antiviral active molecules from the natural product library of traditional Chinese medicine, this study drew on the research ideas of this article, changed the target from EBOV-GP trimer to monomer, and redesigned the experimental method of multi round virtual screening. The screening strategy uses more expensive algorithms to improve the accuracy of prediction as much as possible, reduce the false positive rate of virtual screening, and better predict the binding conformation. In addition, molecular dynamics simulation and binding free energy calculation are also important tools for studying the strength of protein inhibitor interactions. GROMACS software is used for molecular dynamics simulation, and MM/PBSA algorithm is used to predict the binding free energy of the system after stabilization. These methods not only provide details of the interaction between proteins and inhibitors at the atomic and molecular levels, but also facilitate the elucidation of the structural affinity relationship between protein inhibitor complexes.

In order to effectively interfere with the conformational transition of EBOV sgP, this study targets the active cavity of EBOV GP monomer and excavates inhibitor molecules from the natural product library of traditional Chinese medicine to help block host virus membrane fusion. Firstly, follow the Lipinski Five Principles and Verber Three Principles to filter out compounds that violate the principle of drug likeness; Then, Glide software was used to perform molecular docking calculations with three different accuracies, namely high-throughput HTVS, standard precision SP, and ultra precision XP, gradually reducing the size of the candidate compound library; Subsequently, Schrodinger’s QikDrop program was used to predict the ADMET properties of the hit molecule, and compared with admetSAR for filtering strategy. Two key indicators for pharmacokinetic and drug safety analysis, including hepatotoxicity HERG_IC50 and oral bioavailability, were added. Combined with Lipinski Ro5 and Verber Ro3 predicted values, a relatively comprehensive drug like evaluation was made; In the process of analyzing the binding mode between target proteins and hit molecules, high-performance GPU acceleration technology was used to simulate the motion state of biomolecules under real physiological saline solvent conditions. Molecular dynamics simulations were conducted for 50 ns, and MM/GBSA and MM/PBSA algorithms were used to predict the binding free energy and improve enrichment accuracy. Finally, based on the XP Glide score, RMSD, RMSF, MM/GBSA binding energy, MM/PBSA binding energy, and ADME prediction, the hit molecules MOL006834, MOL000174, MOL002192, and MOL012524 showed excellent binding affinity and good drug like properties towards the EBOV sgP active site; Predict the true binding conformations of hit molecules MOL006834, MOL000174, MOL002192, MOL012524 with transmembrane glycoproteins at the atomic and molecular levels, including hydrogen bonding, hydrophobic interactions, ionic bonds, π – π stacking forces, etc., showing excellent interactions with key amino acid residues. These four hit molecules can be commercially purchased and developed as lead compounds for EBOV sgP inhibitors, as well as validated through drug activity experiments. It should be pointed out that Ebola virus has a high mortality rate and high transmissibility, and the relevant biosafety requirements are extremely strict. Antiviral activity testing experiments need to be conducted in biosafety level 4 laboratories, and there are not many laboratories in China with this level, which restricts basic research on Ebola virus. Therefore, the results of this study are limited to the early stages of drug development and lack experimental data support such as antiviral activity validation and ADMET evaluation. In the later stage, it is necessary to collaborate with relevant virology research teams and use professional virology research platforms to carry out safe and effective biological validation.