The Optimization Of LFS-01 As CRM1 Inhibitor And The Study Of Its Derivatives Design And Anticancer Mechanisms

Chromosomal maintenance 1(CRM1)also known as exportin-1(XPO1),widely expressed in many kinds of tumors.CRM1 is the main transporer that transports the intracellular protein bearing the nuclear export signals(NES)from nuclear to cytoplasm.There are 404 proteins can be transported by CRM1,which are recognized and bound by CRM1.Most of the cargo proteins of CRM 1 can be transported into cytoplasm followed by lossing the anti-tumor activity.Therefore,CRM1 plays a critical role in the development of tumor and as one of the hot cancer targets.The serious toxicity and side effects of the only FDA-approved CRM1 inhibitors KPT-330 are the main reseaons of the poor treatments efficacy.There is urgent need to develop noval CRM1 inhibitor with low toxicity.LFS-01(Sulforaphene),which screened from the Chinese Natural Database was proved as an effective low toxicity CRM1 covalent inhibitor.The isothiocyanate group can covalent binding to Cys539 of CRM1 by Micheal addition reaction than blocking the transport function of CRM1.We found that LFS-01 inhibits the TNBC cells proliferation at micro-molar concentrations.Aim to optimizing the activity of LFS-01,we successfully screened out noval inhibitor of LFS-31 based on the integrated approach.Moreover,LFS-1107 was screend out based on the Artificial Intelligence(AI)designed method.The work in this thesis was focus on designing,screening and verifying of the anticancer mechanism of LFS-31,and verified the mechanism of LFS-1107 suppress the progression of BCSC(Breast cancer stem-like cells).1.149 cases of pathological data from breast cancer patients showed that CRM1 was widely expressed and high CRM1 expression patient with poor overall survival.The cell viability of LFS-01 to diverse breast cancer cells was executed by CCK-8 kits.Noteworthy,LFS-01 exhibits a stronger suppression on the growth of the TNBC cell lines(MDA-MB-231 and MDA-MB-468)than non-TNBC(MCF-7 and BT474)cell cines,yet with minimal effects on human mammary epithelial cells.In addition,Western Blot results revealed that LFS-01 can inhibit the transcriptional activity of STAT3(Signal Transducer and Activator of Transcription 3).Immunofluorescence and Western Blot technique further confirmed that LFS-01 induced autophagy.Finally,Western Blot and flow cytometry verified that LFS-01 induced apoptosis.2.At first,a phenol ring was inserted next to the methylsulfinyl bond of LFS-01,leads to a new compound named LFS-06 with the IC50 value of 0.98 μM.The anticancer activity of LFS-06 is 15.9 times higher than LFS-01.For further evaluating the activity of LFS-06,205 substitutes were inserted at the 3,5 position of the phenol ring,then a virtual library with 42025 compounds was build.According to the principle of drug similarity,the drug-likeness of A/DMET,and the molecule docking methods,LFS-31 was selected on the basis of binding energy,drug-likeness properties and easy synthesis.The IC50 value of LFS-31 is 7 times higher than LFS-06,103 times higher than LFS-01.By performing experimental evaluation,the anticancer mechanisms were verified that LFS-31 inhibits NF-κB transcriptional activity by interrupt the binding relationship of IκBα and CRM1,and induces apoptosis.3.Biological evaluation of LFS-1107.LFS-1107 was designed by the artificial intelligence(AI)method.The IC50 value of LFS-1107 to MDA-MB-231 cell is 40.8 nM,which is 24 times higher than LFS-06,300 times higher than LFS-01.And LFS-1107 is more effective than KPT330 to MDA-MB-231 cells.Western Blot assay confirmed that LFS-31 inhibits the expression of p-STAT3.In addtion,the expression of Survivin reached the highest levels in the nuclear following to decrease after LFS-1107 treatment for 3 hours Immunoprecipitation(IP)assay showed that STAT3 binds to Survivin in the nucleus under LFS-1107 treated for 3 hours.Noteworthy,LFS-1107 exhibits high anti-tumor activity with IC50 value from 140 nM to 1.5μM to the PDTO patient model.It was verified that LFS-1107 effectively inhibits the proliferation against TNBC cells in vivo.Noteworthy,we found that LFS-1107 can strongly decreased the cancer stem cells by collecting MDA-MB-231 stem cells through flow cytometry.And we confirmed that LFS-1107 can inhibit the expression of the stem cell hallmarks,such as Sox-2,Oct4,and Nanog by Immunohistochemistry.4.Through the 149 cases of ESCC(esophageal squamous cell carcinoma)pathological data,it is found that STAT3 and NF-κB are over expressed relative to the adjacent tissues,and there is a correlation between the co-expression of NF-κB and STAT3.Western Blot proved that LFS-1107 can significantly dual inhibit STAT3 and NF-κB activity.And by constructing a tumor model of ESCC cells,it is verified that LFS-1107 can inhibit the proliferation of ESCC cells in vivo.In summary,in this thesis we successfully designed a noval CRM1 inhibitor,enhanced the activity,and investigated the anti-cancer mechanism of a series of CRM1 inhibitors.This study provides an important reference for the development and clinical application of CRM1 inhibitors.