Targeted Anti-cancer Drug Discovery By Structure Based Drug Repositioning Strategy

This dissertation consists of two chapters.In Chapter 1,structure-based virtual ligand screening method was employed to screen the ZINC Drug Database to find potential IDH1-mutant inhibitors.The in vitro enzymatic inhibition assay was performed to evaluate inhibitory effects of compounds.Then,the microscale thermophoresis(MST)was used to assess the binding ability between compounds and IDH1R132 H.Finally,clomifene was selected for in vivo antitumor experiment in mice.Chapter 2 discusses the proton pump inhibitors and HJ001 targeted TOPK kinase to inhibit cancer growth.Chapter 1 Structure Based Discovery of Inhibitor of Cancer-Associated Mutant IDH1 Background:Isocitrate dehydrogenases(IDH)are a family of enzymes that catalyzes the conversion of isocitric acid(ICT)to α-ketoglutaric acid(α-KG)which is one of the key reactions in the tricarboxylic acid cycle.Accumulated evidences have revealed mutant IDH enzymes are observed in multiple human cancers.Reportedly,among the three isoforms of IDH in humans,frequent somatic mutations of IDH1 have recently been found in certain cancers including nearly 80% in grade II-III gliomas,appropriate 45% in secondary glioblastoma multiforme(GBM),and 33%-50% in adult primitive neuroectodermal tumor.Besides brain tumors,IDH1 mutations have also been detected in other cancers including acute myeloid leukemia,colorectal cancer.Biochemically,all of the mutant IDH proteins,including IDH1R132 H and IDH1R132 C,concomitant gain of a neomorphic function that reduce α-KG to 2-hydroxyglutaricacid(2-HG)using NADPH as the cofactor.2-HG has been confirmed to be an inhibitor of α-KG-dependent dioxygenases,therefore,as a result of mutations in IDH,high cellular concentration of 2-HG may cause global methylation of histone and DNA,which may lead to tumorigenesis.In summary,these findings suggested that novel specific inhibitors of mutant IDH1 may become a new therapy for glioma,AML and other cancers with IDH1 mutation.Nowadays,structure-based virtual ligand screening is playing an increasingly important role in early-stage drug discovery.Results and discussion:In this study,we conducted a structure based virtual ligand screening to identify small molecule inhibitors of mutant IDH1.Followed by further examination and verification,we confirmed clomifene,an effective and low-cost medication for ovulation induction,as a novel inhibitor of mutant IDH1,which could reduce the cellular and tumor levels of 2-HG.In vivo studies indicated that clomifene significantly suppressed the tumor growth of HT1080-bearing CB-17/Icr-scid mice with oral administration of 100 mg/kg and 50 mg/kg per day.In silico screening of an in-house natural product database and confirmatory bioassays led to the identification of asperspiropene A as an inhibitor of cancer-associated mutant IDH1.Asperspiropene A,a meroterpenoid with a highly functionalized 1,8-dioxaspiro[4.5]decane motif,showed potent ability to inhibit mutant IDH1.Asperspiropene A reduced the cellular concentration of 2-HG in HT1080 cells.Molecularly,treatment with asperspiropene A led to reduced levels of the H3K9me3 signal in the HT1080 cells.To the best of our knowledge,asperspiropene A is the first fungal metabolite reported to exhibit inhibitory activity against mutant IDH1.Three natural steroids from Ganoderma sinense,a unique and rare edible-medicinal fungi in China,were found as potential IDH1 inhibitors by virtual ligand screening method.Among the three compounds,wm-49 showed highest binding affinity to IDH1 with significant calculated binding free energy.Enzymatic kinetics also demonstrated that wm-49 inhibited mutant enzyme in a noncompetitive manner.Wm-49 reduced the cellular concentration of 2-HG in HT1080 cells.The levels of H3K9me3 in HT1080 cells were reduced by treating with wm-49.In short,our findings highlight wm-49 may have clinical potential in tumor therapies as a safe and effective inhibitor of mutant IDH1.Chapter 2 Structure Based Discovery of Inhibitor of T-cell-originated protein kinase Background:TOPK(T-lymphokine-activated killer cell-originated protein kinase)is a Ser/Thr protein kinase,also known as PBK or PDZ-binding kinase,which belongs to the MEK protein family.The PBK/TOPK protein is up-regulated in certain types of cancer cells such as breast,colon,lung and glioma cancer cells,but is difficult to be detected in normal tissues except several fetal tissues and germ cells of the testis.In molecular studies,over-expression of TOPK involves in mitotic checkpoint of tumor cells,cell apoptosis and inflammation.Based on these findings,TOPK has become a novel target for cancer treatment attracting more and more attentions.Some TOPK inhibitors,such as HI-TOPK-032 and OTS964 have been reported in 2012 and 2014,respectively.However,none of them had been in clinical trials because of their toxicities or poor pharmacokinetic properties.Results and discussion:Pantoprazole(PPZ)was identified to be a TOPK inhibitor from FDA-approved drug database by structure based virtual ligand screening.Herein,the data indicated that pantoprazole inhibited TOPK activities by directly binding with TOPK in vitro and in vivo.Inspired by our results of pantoprazole,we speculated that other PPIs may also be developed as TOPK inhibitors.Based on the availability,another six proton pump inhibitors(PPIs)in clinical use were screened against TOPK by virtual ligand screening method,microscale thermophoresis(MST)and cytotoxic activity assay.Ilaprazole,a novel proton pump inhibitor,was found to show potent antitumor activities through the inhibition of TOPK-specific kinase function.HJ001 was also identified as a novel TOPK inhibitor.An in vivo study demonstrated that gavage of HJ001 suppressed tumor growth effectively in tumor-bearing mice.Using SW1990 and ES-2 xenograft model,we found that mice treated with HJ001 survived significantly longer than vehicle treated mice.HJ001 suppressed invasion and metastasis of SW1990 in vivo.Our results suggested that HJ001 is a specific inhibitor of TOPK both in vitro and in vivo that may be a novel therapeutic approach for cancers.