Study On The Mechanism Of Action And The Material Basis Of COVID-19 Treatment In Phytolacca Esculenta Based On Bioinformatics And CADD Technology

ObjectiveWe investigated the mechanism of action and material basis of Phytolacca esculenta fo r the treatment of COVID-19 by bioinformatics and computer-aided drug design techniques（molecular docking and molecular dynamics simulation）and in vitro experiments.Methods1.Bioinformatics-based study of the COVID-19 key gene.（1）COVID-19-related microarrays were obtained with the aid of the GEO gene ban k;differential genes were extracted using the limma package.（2）Differential gene co-expression analysis was performed with the aid of the WGN CA package and Pearson correlation coefficients were used to construct networks and matc h genes associated with COVID-19,highlighting genes with significant associations.（3）WGCNA significant module genes were intersected with upregulated differentia l genes and imported into the STRING database to analyse protein-protein interactions.And use Cyto Hubba plugin based on the MCC algorithm for screening the core targets of the tre atment COVID-19（Hub）.（4）KEGG and GO enrichment analysis of intersecting genes with the help of KOB AS-i and DAVID databases.（5）Validation screening of Hub genes in the validation set using the p ROC package to obtain key genes,and gene GSEA enrichment and correlation analysis between genes fo r individual genes.2.Studies on the role and material basis of key genes targeting COVID-19 in Phytola cca esculenta（1）All the active ingredients of Phytolacca esculenta were collected using the TCM SP database.（2）Molecular docking virtual screening of all active ingredients of Phytolacca escul enta with key genes using Auto Dock Vina software to screen for multiple multi-target comp ounds against COVID-19.3.Spike protein of COVID-19 and ACE2 interactions（1）Protein-protein docking of Spike protein and ACE2 using the ZDOCK server to determine the optimal binding files and binding sites for Spike protein and ACE2.（2）Further molecular docking screening of multi-target compounds with Spike prot eins using Auto Dock Vina software to identify small molecule compounds for potential trea tment of COVID-19 for further studies.4.A computer-level study of the Spike protein-ACE2 interaction targeted by Esculento side A（1）Molecular dynamics simulations using Gromacs software to analyse the binding mode of the complexes of Esculentoside A and Spike proteins.（2）Molecular dynamics simulations using Amber software to analyse the binding en ergies of the complexes of Esculentoside A and Spike proteins and the energy contribution during MD simulations.（3）Absorption,metabolism,distribution,excretion and toxicological properties of E sculentoside A were analysed using Discovery Studio software.5.Study on the in vitro anti-COVID-19 effect of Esculentoside A（1）The drug toxicity of Esculentoside A in HCT-8 cells was examined by MTT ass ay using Ebselen as a positive control.（2）Inhibition of OC43 coronavirus by Esculentoside A was investigated by cytopa thic effect method in different administration models using Ebselen as positive control（3）The compounds screened were assayed for inhibition of main proteinase using e nzyme activity assay with Ebselen as positive control.Results1.Results of bioinformatics-based screening of key COVID-19 targets:（1）A total of 6593 differentially expressed genes were screened through the GEO d atabase,including 3281 up-regulated genes and 3312 down-regulated genes.（2）WGCNA analysis obtained 2 statistically significant modular genes（blue-gree n and blue）that were highly correlated with COVID-9.（3）A total of 1780 identical genes were obtained and 20 Hub genes were screened b ased on the MCC algorithm.（4）GO and KEGG enrichment analysis showed that these targets were involved in 24 signaling pathways,including calcium,P53 and PI3K-Akt signaling pathways as well as s ignaling pathways associated with viral infection such as human T-lymphocyte leukemia vir us type I infection,hepatitis B and hematopoietic cell regulatory cellular pathways.（5）AURKB,BUB1B and ESPL1 genes were upregulated in both the experimental a nd validation sets of NCCP samples.ROC curve analysis showed that the AUC values of al l three genes in the experimental and validation sets were greater than 0.70,which had a hig h diagnostic value.6.The single gene Gsea enrichment analysis showed that the key genes were positively correlated with multiple immune-related pathways（P<0.05）,and the correlation heat m ap demonstrated that all three key genes were positively correlated.2.The results of targeting COVID-19 key genes in Phytolacca esculenta:（1）There were 49 active ingredients in Phytolacca esculenta.（2）A virtual screening of the active components of Shangluo with 3CLpro,AURK B,BUB1B and ESPL1 key target molecules resulted in the potential anti-COVID-19 active compounds Shangluo C,Shangluo saponin A,galic acid and hesperidin with significant doc king effects.3.Results of the interaction between Spike protein and ACE2:（1）The results of multiple crystal junction docking of Spike proteins with ACE2 sh owed that a single S protein（6XR8）had the most interacting residues with the ACE2（1R42）receptor,with 21 and 24,respectively.（2）The Spike protein was molecularly docked again to the potential compound,and the best binding energy of-13.1kcal/mol was obtained for the S-protein by Esculentoside A.（3）The contribution of Esculentoside A-S protein ability showed van der Waals’in teraction,hydrogen bonding,desolvation energy of-24.19 kcal/mol and electrostatic energy of-5.485 kcal/mol.A total of six hydrogen bonds were formed,indicating that hydrogen bo nding plays an important role in the binding of Esculentoside A to S protein.4.Molecular kinetic and pharmacokinetic results of Esculentoside A:（1）The RMSD values fluctuated from 0.218 nm to 0.461 nm,indicating a very stab le structure.The RMSF showed that no significant conformational changes were observed in the active site of the protein and that the binding residues to the S.saponin A were very sta ble.（2）The Rg of the complex system was very stable over the course of 100 ns,between0.2484 and 2.659 nm.（3）Esculentoside A forms hydrogen bonds with Arg594,Pro595,Tyr141,Asn142and Arg238 and also forms critical hydrophobic interactions with Asp592,Trp593,Tyr596,Ser237,Ala597,Arg238,Leu145,Val474,Tyr596 and Ser237 in the active pocket to stabili ze it in the active pocket.（4）the secondary structure is stable in the bend form,the presence of the ligand stab ilises the secondary structure of the S protein but also causes changes in the secondary struc ture of the residues.the total solvent accessible surface area of S protein-Shanglu saponin A is 263.125～301.527 nm and is relatively stable during the simulations.（5）The binding energy of S-protein to Esculentoside A was-13.12 kcal/mol,with r esidue TRP503 contributing the most to the binding energy（<-4.0 kcal/mol）.The major residues near the active pocket,Trp593,Ser240,Pro241,Arg238,Tyr505,Asn501,Gln498and Thr500,play an important role in the binding of the inhibitor to S-protein.（6）PCA analysis demonstrated that the movement of the complex system was insig nificant during the simulation and that the structure of the Spike protein and the Esculentosi de A complex was relatively stable.（7）Pharmacokinetics indicate that Esculentoside A is a safe compound with poor b ioavailability and low toxicity and carcinogenicity,and is a fat-soluble drug with good abso rption.5.Esculentoside A has HCOV-OC43 effect on HCT-8 cells in an in vitro assay（1）On HCT-8 cells,the CC₅₀ of Esculentoside A semitoxic concentration was 106.6μM,and the CC₅₀ of ebselenoside was 82.23μM.（2）The IC₅₀ was 9.278μM and 33.56μM,and the TI indices were 12.5 and 3.36 for HCOV-OC43 in the prophylactic and therapeutic modes,respectively,but the pre-mixed gr oup did not show antiviral effects and the TI indices were<1.（3）In the main protease activity assay,Esculentoside A showed 24.16%inhibition a t 40μM with an IC₅₀ of 267.5 u M.ConclusionThe small molecule compound Esculentoside A was identified by computer-aided drug design methods such as bioinformatics,molecular docking,molecular dynamics simulation and pharmacokinetic prediction,and Esculentoside A it was able to bind stably to S protein s and form hydrogen bonds,in addition,it also showed good pharmacokinetic properties.Es culentoside A showed in vitro（HCT-8）anti-HCOV OC43 effect and anti-SARA-COV-2main protease activity.Combining the results of both computer and in vitro experiments,it was found that Esculentoside A could act on multiple targets and modulate multiple signalin g pathways for the treatment of COVID-19,and has the potential to be developed as an anti-SARS-COV-2 virus drug.