| Object:Prostate cancer and esophageal cancer are common malignant tumors worldwide,and their incidence and mortality have increased year by year,which seriously threaten human health.Peptidyl-prolyl cis-trans isomerase Pin1 can specifically recognize and bind to the pSer/Thr-Pro site and catalyze the cis-trans isomerization of phosphorylated proteins containing this site,regulating protein activity,protein stability,Protein-protein interactions and subcellular localization.Pinl is overexpressed in many tumor cells and tissues,such as prostate cancer,esophageal squamous cell carcinoma,breast cancer,lung cancer,and melanoma.Pinl activates more than 50 oncogenes or proliferative factors while inhibits more than 20 tumor suppressor genes or anti-proliferative factors,and can regulate multiple cancer-driven pathways at the same time,playing a key role in tumor progression,and closely related to clinical stage and prognosis.Therefore,Pinl is a good biomarker and prognostic indicator in tumors,and it is the current research focus and direction.In recent decades,many small molecule inhibitors targeting Pinl have been developed through high-throughput screening and rational design based on mechanism and structure.Although they reach low micromolar or low nanomolar levels at the enzyme level,their cell permeability is poor,and they are inactive or less active at the cellular level.Therefore,it is urgent to find a small molecule inhibitor that targets Pinl with high efficiency and good cell permeability.This project intends to screen a highly effective and highly permeable compound targeting Pinl through virtual screening and Western Blot,to study the antitumor activity of the compound in vitro,and to explore its mechanism of action.Methods:In this experimental study:(1)Western blot was used to determine Pinl overexpressing tumor cell lines for subsequent experiments.(2)Leading compounds were obtained by computer-assisted virtual screening and cell-level activity evaluation,and a series of derivatives were optimized based on the structure of the compound,and a small molecule inhibitor 1169 that significantly inhibited Pinl activity was obtained through further cell-level screening.(3)Transient transfection was used to determine the effective Pinl interference plasmid,and this plasmid was used to construct Pinl stably silenced cells.(4)Through thermal migration and Co-IP,and detecting the expression of Pinl and its downstream proteins in stable shPinl transgenic plants treated with compound 1169,the targeting activity of compound 1169 at the cell level against Pinl was determined.(5)In PC3,KYSE70 and shPinl cells,MTT,clone formation assay,transwell and detection of migration-related protein expression were performed to evaluate the effect of compound 1169 on cell proliferation,clone formation,and migration,and its targeting to Pin1 Inhibition.(6)The effect of compound 1169 on the cycle of PC3 and KYSE70 cells was detected by flow cytometry.(7)The effect of compound 1169 on the downstream protein expression of Pin1 was detected by Western blot,and the mechanism of compound 1169 in PC3 and KYSE70 cells was preliminarily explored.(8)The mice were given a high dose of compound 1169 by gavage.After 2 weeks,the main organs(heart,liver,spleen,lung,kidney)were obtained,and paraffin embedding,sectioning and HE staining,and the toxic and side effects of compound 1169 on mice were initially determined.Results:(1)Among 11 kinds of tumor cells and 2 kinds of normal cells,the expression level of Pinl in prostate cancer cell PC3 and esophageal cancer cell KYSE70 was higher and significantly higher than that in normal cells.(2)Based on the crystal structure of Pinl,computer-assisted virtual screening was used to obtain 30 compounds,and the effect of compounds at the cellular level on the expression of CyclinD1,a downstream protein of Pinl,was detected to obtain a lead compound 12 that significantly inhibited Pinl activity.(3)In Pin1 overexpressing prostate cancer cell PC3 and esophageal cancer cell KYSE70,compared with other derivatives,compound 1169 significantly inhibited the expression of CyclinD1,β-catenin,NFκB,and AKT downstream protein of Pin1.Therefore,the compound 1169 was screened to significantly inhibit Pinl activity.(4)An effective Pinl interference plasmid shPin1-835 was obtained,and PC1 and KYSE70 stably transfected cell lines were successfully constructed.(5)In PC3 and KYSE70 cells,CETSA results showed that compound 1169 bound to Pin1 at the cellular level,increasing the thermal stability of Pinl;Co-IP results showed that compound 1169 significantly inhibited the binding between Pinl and CyclinD1;After compound 1169 treated shPin1 cells,there was no significant difference in the expression of Pin1 downstream proteins in shPin1 group and shPin1+1169 group,indicating that compound 1169 interacted with Pin1.The above results collectively show that compound 1169 targets Pinl in PC3 and KYSE70 cells.(6)In PC3 and KYSE70 cells with high Pin1 expression,the inhibitory effect of compound 1169 on cell proliferation,colony formation,and migration was consistent with the results of Pinl silencing,indicating that compound 1169 significantly inhibited cell proliferation,colony formation,and migration by targeting Pin1.(7)The cell cycle results showed that with the increase of the compound 1169 concentration,the G0/G1 phase ratios in PC3 cells were 63.71%,71.22%,76.73%,and 83.02%(P<0.01),and the G2/M phase ratios in KYSE70 cells were 12.28%,14.12%,14.54%,and 23.73%(P<0.01).It showed that compound 1169 significantly caused G0/G1 phase arrest in PC3 cells,and G2/M phase arrest in KYSE70 cells,and had cell specificity.(8)Compound 1169 significantly down-regulated the expression of CyclinDl,β-catenin,NFκB,and AKT of the Pinl downstream proteins in PC3 and KYSE70 cells.The compound may reduce the stability and expression level of Pin1 downstream proteins CyclinD1、β-catenin、NFκB、and AKT by inhibiting Pinl activity,thereby showing an antitumor effect.(9)2g/kg of compound 1169 did not cause mouse death,did not affect the normal growth of mice,and did not cause significant damage to important organs in mice.Therefore,compound 1169 has good safety in vivo.Conclusion:In this study,through computer-assisted virtual screening and cell-level activity evaluation,we obtained a compound 1169 that targets Pin1 and has good permeability in Pinl overpressing PC3 and KYSE70 cells.Compound 1169 significantly inhibited cell proliferation,colony formation,and migration,causing cell cycle arrest and inhibiting the expression of Pinl downstream proteins in a concentration-dependent manner.The antitumor mechanism of compound 1169 is related to its inhibition of Pin1 activity,which reduces the stability and expression levels of Pin1 downstream proteins CyclinD1、β-catenin、NFκB、and AKT.At the same time,compound 1169 has good safety in vivo and can be used for subsequent structural optimization and in vivo activity evaluation studies. |
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