Chemotherapy targeting human thymidylate synthase messenger RNA

描述（由申请人提供）：拟议的研究旨在发现抑制胸腺苷酸合成酶（TS）表达的新型抗癌药物，TS是用于DNA生物合成的胸腺苷酸的唯一细胞内新生来源。具体来说，我们希望合成并鉴定能够选择性结合并稳定TS mRNA 5'非翻译区（NTR）结构调控元件的化合物，该区域包含隔离在RNA发夹元件中的翻译起始密码子。通过阻止细胞翻译机制进入起始位点，TS mRNA结合化合物将模仿TS蛋白的作用，TS蛋白作为其自身mRNA的配体和阻遏物。在初步实验中，我们已经通过稳定mRNA二级结构验证了TS mRNA作为翻译抑制的靶标。在拟议的研究中，我们将建立一个迭代过程，旨在发现先导化合物，用于开发新的癌症治疗药物，与现有的抗癌药物联合使用，这些药物靶向该酶，但通过上调TS表达而产生耐药性。本课题的具体目标是：1)设计和合成基于两类有利于RNA结合的杂环的新型RNA偏配体；2)设计并验证代表TS mRNA 5'-NTR中调控RNA元件的模型寡核苷酸，用于配体结合研究；3)通过x射线晶体学确定ts5′- ntr中调控RNA元件的三维结构；4)建立对TS mRNA的RNA亲和力和靶特异性分析，在新合成的RNA偏向配体和已知的RNA结合天然产物中鉴定小分子结合物；5)评估TS mRNA结合物在结合和体外翻译实验中的特异性靶识别能力；6)在人细胞系中检测TS特异性翻译抑制剂，测定其细胞膜通透性、对TS表达的干扰及抗增殖活性；7)通过x射线晶体学确定TS mrna -配体复合物的三维结构；8)利用生物活性数据和结构信息（结构活性关系，SAR）设计具有潜在改善结合亲和力的修饰配体。

DESCRIPTION (provided by applicant): The proposed research is intended to discover novel anti-cancer agents that inhibit expression of thymidylate synthase (TS), the sole intracellular de novo source of thymidylate for DNA biosynthesis. Specifically, we want to synthesize and identify compounds that selectively bind and stabilize a structured regulatory element in the 5' nontranslated region (NTR) of the TS mRNA which contains the translation start codon sequestered in an RNA hairpin element. By preventing access of the cellular translation machinery to the initiation site, TS mRNA-binding compounds would mimic the action of TS protein which acts as a ligand and repressor of its own mRNA. In preliminary experiments we have validated the TS mRNA as a target for translation inhibition via stabilization of the mRNA secondary structure. In the proposed research we will establish an iterative process aiming at the discovery of lead compounds for the development of novel cancer therapeutics for use in combination with existing anti-cancer drugs that target the enzyme but suffer from resistance development via upregulation of TS expression. The specific aims of this project are to: 1) design and synthesize novel RNA-biased ligands based on two classes of heterocycles privileged for RNA binding; 2) design and validate model oligonucleotides representing the regulatory RNA element in the 5'-NTR of TS mRNA for ligand binding studies; 3) determine the three- dimensional structure of the regulatory RNA element in the TS 5'-NTR by X-ray crystallography; 4) develop RNA affinity and target specificity assays for the TS mRNA and identify small molecule binders among the newly synthesized RNA-biased ligands and known RNA-binding natural products; 5) evaluate TS mRNA binders for specific target recognition in binding and in vitro translation assays; 6) test TS-specific translation inhibitors in human cell lines to determine membrane permeability, interference with TS expression and antiproliferative activity; 7) determine the three-dimensional structure of TS mRNA-ligand complexes by X-ray crystallography; 8) design modified ligands with potentially improved binding affinity by using biological activity data and structural information (structure activity relationships, SAR). PUBLIC HEALTH RELEVANCE: This research is aimed at the discovery of novel anti-cancer therapeutics which will greatly increase the efficacy of existing drugs for the treatment of resistant breast and colorectal cancer.