Screening,Validation And Preliminary Mechanism Study Of Small Molecule Inhibitors Of STAT3 Protein In Pancreatic Cancer

[Objective]Due to the effect of various adjuvant therapies is limited and the prognosis is extremely poor,the increasing incidence and mortality of pancreatic cancer year by year.Therefore,there is an urgent need to innovate its treatment.STAT3 protein plays an important role in the progression,metastasis,immunosuppressive microenvironment formation and treatment resistance of pancreatic cancer.Exploring more compounds with anticancer activity that directly target STAT3protein into preclinical research and clinical transformation will improve the prognosis of pancreatic cancer patients.[Methods]Firstly,we analyzed the baseline data of clinical cases,immunohistochemical staining and proteomic analysis of tumor tissues,prognostic data analysis,and bioinformatics analysis,to clarify the important role of STAT3 in the clinical prognosis,pathogenesis and progression of pancreatic cancer patients and the changes of intracellular signaling pathways.Based on the computer simulation molecular docking technology,the ZINC-Lead-Like small molecule compound database was screened by Schrodinger software targeting the SH2 domain of STAT3protein,and the candidate compounds were verified by Auto Dock Vina software.Candidate compounds were screened by CCK-8 assay,scratch migration assay,Transwell invasion assay,RT-PCR and WB,for inhibitory effect on STAT3 protein function and anti-pancreatic cancer activity.Then,SPR,CETSA,CCK-8 assay,flow cytometry,RT-PCR and WB were used to further clarify the anti-pancreatic cancer effects and their mechanisms of action of the main candidate compounds.The key candidate compounds were selected and injected intraperitoneally into the pancreatic cancer orthotopic CDX mouse model.The tumor histopathology,cardiac,hepatic and renal toxicity,drug mechanism and macrophage markers were further observed by measurement,HE staining,TUNEL,immunohistochemistry and immunofluorescence.[Results]（1）p-STAT3 was strongly elevated in pancreatic cancer compared to healthy pancreatic tissue（p<0.05）,and the expression was positively linked with a poor prognosis（p<0.05）and M2 macrophage infiltration（p<0.05）.According to DIA proteomics,the PI3K-AKT signal pathway,mismatch repair,DNA replication,homologous recombination,VEGF signal route,pancreatic cancer,cell cycle,and Ras signal pathway are where the roles of p-STAT3-related differential proteins are most clearly seen.Bioinformatics studies also suggested that the prognosis of pancreatic cancer patients with high expression of STAT3 mRNA was worse,and STAT3 was positively correlated with IL-6,JAK2,VEGF family（especially VEGF1）,factors related to EMT pathway,cell cycle regulation and macrophage infiltration（p<0.05）.（2）50 small molecular compounds with binding energies of-10.056 to-12.485kcal/mol were virtually screened by Schr（?）dinger software,and the results of Autodock Vina software（binding energy-5.2 to-6.8 kcal/mol）also suggested that the top ten compounds with binding energy（compounds 1-10）and BA interacted with STAT3 protein.Specifically,the function of STAT3 protein is inhibited by forming more hydrogen bonds and hydrophobic interaction with SH2 domain.（3）Compound 1’s IC50 was calculated at 12 hours（27.302 M）and 24 hours（23.013 M）.In pancreatic cancer PANC-1 and Bx PC-3 cells,all candidate medications（compounds 1–10 and BA）demonstrated varying degrees of proliferation suppression,considerable invasion inhibition（p<0.05）,and significant anti-migration effects（p<0.05）,with the exception of compound 6 and compound 7（p<0.001）.The phosphorylation of STAT3 and JAK2 proteins in pancreatic cancer PANC-1 and Bx PC-3 cells could be considerably inhibited by all prospective medicines（p<0.05）.Comparatively,it was discovered that compound 4(The following are Chem Div^TM ID:Y200-4277),compound 5（Y204-0240）,and compound 9（4207-4272）had the most pronounced alterations out of all the medication candidates.（4）The 24h IC50 of compound 4（5.721μM/5.487μM）,compound 5（5.812μM/5.768μM）and compound 9（14.535μM/14.488μM）on PANC-1 and Bx PC-3 cells was determined,and it was found that compound 4（KD=8.58E-05 M）,compound 5（KD=2.15E-04 M）and compound 9（KD=2.44E-04 M）could bind to humanized STAT3 protein at moderate-high intensity.CETSA assay showed that compound 4,compound 5 and compound 9 could enter into pancreatic cancer cells and bind to intracellular STAT3 protein.In addition,three compounds had no significant effects on STAT1 protein and STAT5 protein.（5）Compounds 4,5,and 9 all increased apoptosis（p<0.001）,stopped the G0/G1 phase（p<0.001）,and decreased the proliferation（p<0.001）of PANC-1 and Bx PC-3 cells in a concentration-dependent manner.With the increase of drug concentration,the expression of Ki67 and PCNA decreased（p<0.001）,the expression of BCL-2 decreased,the expression of BAX and Cleaved Caspase-3increased（p<0.001）,the phosphorylation of STAT3 protein was inhibited（p<0.001）,the phosphorylation of JAK2 protein and IL-6 protein decreased（p<0.001）,the phosphorylation of CDK2 protein was inhibited（p<0.001）,and the expression of VEGF protein decreased（p<0.001）.The expression of E-Cadherin protein increased（p<0.001）,the expression of Vimentin and Snail decreased（p<0.001）,the level of STAT3 and JAK2 mRNA increased synchronously（p<0.001）,but the level of IL-6mRNA decreased（p<0.001）.（6）After in vivo administration of compound 4 and compound 5,compared with blank group（0.92±0.11g）and solvent group（0.88±0.07g）,compound 4 high（0.22±0.14g）,medium（0.48±0.24g）,low（0.54±0.10g）concentration group and compound 5 high（0.23±0.07g）,medium（0.42±0.11g）,low（0.64±0.16g）concentration groups significantly decreased the tissue quality of pancreatic tumor in situ（p<0.001）.HE staining showed that the atypia of tumor tissue,cell polymorphism and neovascularization decreased after administration,and there was no toxic effect of heart,liver and kidney after administration.With the increase of drug concentration,STAT3 protein phosphorylation was inhibited,Ki67 expression decreased（p<0.001）,TUNEL fluorescence expression increased（p<0.001）,IL-6 and p-JAK2 expression decreased（p<0.001）,p-CDK2 and VEGF expression decreased significantly（p<0.001）,E-Cadherin expression increased（p<0.001）,Snail expression decreased（p<0.01）,and CD86/CD163 ratio increased（p<0.05）.[Conclusions]STAT3 has a significant role in the occurrence and progression of pancreatic cancer.The clinical prognosis of individuals with pancreatic cancer may be significantly improved by inhibiting the STAT3 protein’s function.Compounds 1-10and BA screened based on computer-assisted molecular docking technique can bind to the SH2 domain of human STAT3 protein and inhibit its function,so they could be used as candidate drugs for small molecular inhibitors targeting STAT3 protein.Among them,compound 4,5 and 9 have the strongest anti-tumor effects,that can prevent two pancreatic cancer cell lines from proliferating,migrating,invading,and targeting inhibit JAK2/STAT3 protein.Compounds 4,5 and 9 can target and prevent the phosphorylation of the STAT3 protein in pancreatic cancer cells,restrict the activity of the downstream CDK2,VEGF,and other critical factors,coordinate the regulation of proliferation-and apoptosis-related proteins,and control the EMT pathway.Ultimately,these compounds can prevent the proliferation,migration,and invasion of pancreatic cancer cells and promote cell cycle arrest.Furthermore,in vivo,compounds 4 and 5 have anti-tumor activity and safety,and they may mediate the changes of immune microenvironment and intracellular molecular changes to exert anti-tumor effect.