Virtual Screening Of Histone Deacetylase Inhibitors And Its Anti-tumor Molecular Mechanisms

Histone Deacetylases(HDACs)can remove the negatively charged acetyl functional groups from histone or protein lysine residues,resulting in enhanced electrostatic interaction between histones and DNA.It will inhibit the expression of tumor suppressor genes,lead to proliferation of tumor cells and inhibit apoptosis of tumor cells.While the Histone Deacetylase Inhibitor(HDACi)can reduce the chromatin structure by inhibiting the deacetylation of histones,which is more conducive to the binding of transcription factors,so it play an important role in regulating the expression of oncogenes.At the same time,HDACi can also regulate the acetylation level of some non-histone proteins and affect the expression of genes by affecting the stability of their proteins.Lots of studies have shown that HDACs are closely related to various tumors,even though five HDACis have been approved by FDA for clinical treatment,but these drugs also have some side effects,such as thrombocytopenia,gastrointestinal discomfort and arrhythmia.Therefore,the search for new and more effective HDACi has always been an important direction for the development of anti-tumor drugs.In addition,the mechanisms of the anti-cancer effects of HDACi has not been fully elucidated,which is also an important direction for the development of antitumor drugs.In our study,HDACs were used as target to screen for new HDACi compounds by virtual screening methods,and further analysis its anti-cancer effects and clarify its anticancer molecular mechanisms.The main contents of our research are as follows:1.Virtual screening for new HDACiHere,we improved the virtual screening process,using SYBYL pharmacophore method and GOLD molecular docking method to screen the ZINC database,and then use the Selector module in SYBYL-X 2.0 software to cluster the structure diversity of the selected small molecules.Then,25 compounds with different structures are obtained.Subsequently,we use AMBER18 to do 2 ns molecular dynamics simulation of HDAC8 with compounds and the results of the last 1 ns were selected for calculating the bound free energy.Finally,according to the results of computer virtual screening,13 hit compounds were selected for the next biological experiments.In vitro enzymatic activity experiment indicated that ZINC24469384,ZINC48302525 and ZINC08671161 can significantly inhibited HDACs.Anti-cancer experiments indicated that ZINC24469384 can inhibit the activity of liver cancer cells(HepG2,Hep3B),lung cancer cells(A549),ovarian cancer cells(A2780)and gastric cancer cells(SGC-7901)but the dose have non-toxic to normal cells(L02).And ZINC24469384 can obviously inhibite the activity of liver cancer cells.The results of flow cytometry analysis showed that the percentage of apoptotic cells and G1/S phase cells were significantly increased after ZINC24469384 treated in liver cancer cells.Western Blot results indicated that the expression of apoptosis associated protein Bax was significantly increased,while the expression of Bcl-2 was down-regulated,and the expression of cell cycle-associated proteins CyclinA and CyclinB was significantly inhibited.These results indicate that we have obtained a new benzamide HDACi ZINC24469384,which hit by a series of virtual screening methods and it can inhibit the cell activity of many cancer types,expecially the liver cancer cells.2.The molecular mechanism for anti-cancer effect of ZINC24469384In order to further analysis the molecular mechanism of anti-cancer effects of ZINC24469384,we first using next-generation sequencing methods to detected the change of genes expression in HepG2 cells after 4,16 and 24 hours of ZINC24469384 treated,then analyzed the DEGs and DASGs.The DEGs pathway enrichment results indicated that ZINC24469384 can cause changes in FXR/RXR pathway,JAK/STAT pathway,p53 signaling pathway and apoptotic pathway,DASGs can also cause changes in the p53 signaling pathway and cell cycle pathway.The results indicate that DEGs and DASGs can participate in some signaling pathways respectively or together to contribute to the antitumor effect of ZINC24469384.In addition,ZINC24469384 can also induce the expression of liver-specific expression gene NR1H4 in FXR/RXR pathway,then induce the expression of SOCS2 in the JAK/STAT pathway and inhibit the phosphorylation of STAT3.These results also confirmed by qRT-PCR and Western-blot.These results indicated that activation of NR1H4/SOCS2 may be one of the mechanisms by which ZINC24469384 specifically inhibits the viability of liver cancer cells.To further validate our conclusions,we analyzed the necessity of NR1H4/SOCS2 in the anti-cancer effects of ZINC24469384.Western blot,qRT-PCR and flow cytometry analysis revealed that ZINC24469384 can not induce the expression of SOCS2,phosphorylation of STAT3,cell cycle arrest and apoptosis of cells when we interfered with the expression of NR1H4 in HepG2 cells.Therefore,activation of NR1H4 is a necessary gene for ZINC24469384 specificlly inhibit the activity of HepG2 cells.At the same time,the mRNA expression data and clinical stage data of liver cancer(LIHC)and cholangiocarcinoma(CHOL)in TCGA database showed that the expression of NR1H4 and SOCS2 was downregulated with the increase of malignant degree of tumor,indicated that NR1H4 and SOCS2 may be involved in tumorigenesis.In summary,we have hit a novel benzamine lead compound of histone deacetylase inhibitor ZINC24469384 from ZINC database,which may specifically inhibit the activity of liver cancer cells by activating the expression of NR1H4.This study provides a new field for the design of HDACi,and provides a solid theory foundation for the study of the anticancer mechanism of HDACi.