Anticancer Drug Discovery And Activity Evaluation Of Targeting Glutamic Oxalamide Transferase(GOT1)

This thesis consists of two chapters.The first chapter is about the discovery process and activity evaluation in vitro of natural product inhibitor Aspulvinone O against Glutamic-oxaloacetic transaminase 1(GOT1).The second chapter principally discusses the screening of GOT1 inhibitors from approved compound library and the study on the mechanism of anti-tumor activity.Chapter 1Pancreatic ductal adenocarcinoma(PDAC)cells relies on a non-canonical and distinct pathway in which glutamine-derived aspartate can be converted into oxaloacetate catalyzed by aspartate transaminase(GOT1)to meet nutrient requirements for sustained proliferation and metabolism.Meanwhile,GOT1 play an important role in energy metabolism and ROS balance.Studies showed that down-regulation of GOT1 in different cell would inhibit PDAC cells’ proliferation while has no influence on normal tissues and cells.To obtain novel GOT1 inhibitors,we evaluated a series of inhouse natural products against GOT1 activity,and then we chose Aspulvinone O(AO)as a research object for its higher activity.In silico analysis revealed that AO bound to GOT1 by forming obvious hydrophobic interaction with Trp141 together with hydrogen bonds with Thr110 and Ser256.The Microscale thermophoresis(MST),drug and target affinity stability experiments(DARTS)and thermal migration experiments(CETSA)further demonstrated the specific binding of GOT1 to compound AO at molecular and cellular levels.Additionally,silencing of the GOT1 gene in several PDAC cell lines and CETSA results have further validated that AO has a higher affinity to GOT1.Furthermore,we demonstrated that AO could inhibit GOT1 by detecting the expression level of metabolites in Glutamine metabolism signaling pathway,and further block the whole signaling pathway.More importantly,AO can significantly inhibit proliferation of SW1990 cell in CB17/SCID mice model in vivo.Taken together,this research presented a novel GOT1 inhibitor which could be regarded as a potential drug candidate for PDAC cancer therapeuticsChapter 2Transforming metabolism to meet the nutritional requirements of tumor microenvironment changes is a hallmark feature of tumor cells.Increasing evidences indicate that various tumor cells show great addiction to glutamine aside from glycolysis.Given the previous study on the GOT1 inhibitor in the metabolic pathway,in order to save the cost of drug development and shorten the time for the drug to enter the clinical experiment,in this stage of the experiment,according to our established the enzyme activity screening system,we perform a screening from our established approved drug library.The screening results showed that the two approved drug,Ziprasidone hydrochloride and Adapalene,could significantly inhibit GOT1 enzyme activity,which were clinically used for the treatment of psychosis and skin acne.In the follow-up study,MTT was first tested for its inhibitory effect on existing tumor cell lines,and its significant inhibitory effect on SW1990 cells and ES-2 cells and these two cells were used as follow-up research priorities.Subsequently,the binding affinity between the compound and the GOT1 protein was demonstrated by MST and DARTs experiments.Meanwhile,the EdU,Hoechst33258,scratch assay,and Transwell migration assays further demonstrated that the two compounds could induce tumor cell apoptosis and inhibit tumor cell proliferation and migration in vitro.More importantly,the CB17/SCID mouse tumor-bearing experiment demonstrated that ziprasidone hydrochloride inhibited the growth of tumor cells in vivo,and the liver immunohistochemistry experiment fully confirmed that salt ziprasidone can inhibit the metastasis of SW1990 cells in vivo.To sum up,we have demonstrated the inhibition of tumor cells of two approved compounds in vivo and in vitro.This "drug repurposing" strategy has great significance for the discovery of inhibitors of tumor cells.