The Novel Small Molecule Targets STAT-SH2D To Suppress Pancreatic Cancer

Pancreatic cancer is one of the most aggressive and worst prognosis malignant tumors.It carries a dismal 5-year survival rate of less than 9%and seriously harms to human health.In the United States,the incidence and mortality rates of pancreatic cancer rank 10th and 4th among all male malignancies,respectively,and in all-female malignancies,they rank 9th and 4th,respectively.In addition,in China,the incidence and mortality rates of pancreatic cancer are also on the rise,ranking 9th and 6th among all tumors in 2015 with the characteristics of more urban patients than rural patients and more elderly patients than young patients.Furthermore,pancreatic cancer is difficult to be diagnosed in the early stage,and it is usually found in the middle and late stage,with a high rate of liver and lymph node metastasis,and resists to the existing treatment strategies,and finally falls into an intractable dilemma.Therefore,it is urgent to develop new drugs for the treatment of pancreatic cancer.Signal transduction and transcriptional activation protein 3(STAT3)is closely related to cell proliferation,differentiation,and apoptosis,which is activated in a variety of tumors.Overexpression and persistent activation of STAT3 is associated with pancreatic tumor progression.STAT3 activation is dependent on tyrosine 705(Tyr705)and serine 727(Ser727)phosphorylation.STAT3-Tyr705 phosphorylation regulates the classical signaling pathway that regulates the transcriptional functions in the nucleus.While,phosphorylation at Ser727is dominated for its non-classical signaling pathway,such as controlling STAT3 to entry into mitochondria and regulating mitochondria oxidative phosphorylation.Besides,the STAT3-SH2 domain is the critical target for the development of direct inhibitors of STAT3,and there are small molecule inhibitors or antibodies that inhibit STAT3 activities by directly binding to the STAT3-SH2 domain.While,the micromolar level of inhibitory activities limits its further applications.Therefore,the screening and development of potent and highly selective small-molecule inhibitors targeting the STAT3-SH2 domain have important academic value and application prospects.We designed and synthesized a series of potential STAT3 small molecule inhibitors through computer-aided drug design and structured drug design strategies,and we have screened and obtained two novel STAT3 inhibitors,WB436B and WB737,by direct binding assays,cellular and in-vivo experiments.WB436B and WB737 have a distinct effect on STAT3 phosphorylation.WB436B inhibits STAT3-Tyr705 phosphorylation,while,WB737inhibits STAT3 Tyr705 and Ser727 phosphorylation.WB436B selectively binds to the STAT3-SH2 domain among all members of the STAT family.The docking,site-directed mutagenesis and direct binding experiments confirmed the residues of STAT3-SH2D that WB436B bound to.In cellular experiments,WB436B inhibits STAT3-Tyr705phosphorylation and selectively suppresses high-STAT3-Tyr705 level pancreatic cancer cells growth.In the animal models of pancreatic cancer,WB436B is well-tolerated,and potently inhibits the tumor growth and metastasis,significantly prolongs the survival time of tumor-bearing mice.The STAT3 biphosphorylation inhibitor WB737 is co-targeting STAT3 Tyr705 and Ser727 phosphorylation.Our research revealed that WB737 directly binds to the STAT3-SH2 domain,with an affinity(K_D)of 2.27 n M.It simultaneously inhibits STAT3 Tyr705 and Ser727 phosphorylation,suppresses STAT3 classic nuclear function and the non-classical functions,such as mitochondrial oxidative phosphorylation.In cellular experiments,WB737 inhibits pancreatic cancer cell proliferation and induces cancer cells apoptosis within 1-10 n M.In animal models of pancreatic cancer,WB737 inhibits pancreatic cancer growth and metastasis,prolongs the survival of tumor-bearing mice.Besides,the Tyr705 and Ser727 phosphorylation of STAT3 is also suppressed in vivo.In summary,we screened and validated two novel STAT3 inhibitors,WB436B and WB737.WB436B mainly inhibits the STAT3 Tyr705 phosphorylation and its nuclear transcriptional activity,while,WB737 inhibits the STAT3 Tyr705 and Ser727phosphorylation,then suppresses STAT3 nuclear and mitochondrial functions.They significantly inhibit tumor growth and metastasis,and suppress STAT3 activities in vivo and in vitro.Therefore,this study not only evaluates the potential value of two STAT3 inhibitors for the treatment of pancreatic cancer,but also provides new strategies to develop STAT3mono-or dual-site phosphorylation inhibitors.