以原癌基因RET启动子区G4-DNA为靶标的小分子化合物筛选

The RET oncogene encodes a receptor-type tyrosine kinase that plays an important role in the initiation and progression of several human cancers. RET was found to be overexpressed in breast cancer and it has been investigated as a potential therapeutic target in preclinical approaches for the treatment of RET-associated cancers. However, most RET inhibitors are not RET-specific, while they were originally investigated for other tyrosine kinases including VEGF2, MET and ALK etc. The human RET promoter has three GC boxes corresponding to three Sp1 binding sites in the proximal promoter region. Two of these GC boxes, locating between -59 and -25 upstream of the transcription start site, are essential for functional transcription activity of RET promoter. This proximal promoter region containing five guanine tracts forms a stable parallel-stranded G-quadruplex in the presence of K+, a high-order DNA structure, which is related to the gene regulation. It has been proposed that chemicals, which induce or stabilize G-quadruplex structure, can regulate gene expression. The ligands to G-quadruplex in RET promoter are expected to decrease RET expression and further affect cancer cells growth. In this project, we will focus on small molecules screening for RET G4-DNA, and evaluate their binding specificity and biological activities..Firstly, we will do molecular docking operation to virtual screen small molecules which can interact with RET G4-DNA. Secondly, one-dimensional NMR 1H spectra of RET G4-DNA will be analyzed to detect the interactions between RET G4-DNA and small molecules by observing the chemical shifts changes of imino protons of guanines. Thirdly, we will probe the effects on the stability of RET G4-DNA by small molecules through measuring melting temperature using DSC assay, and investigate the possible changes in parallel-stranded folding resulted by small molecules by CD spectra. Then, NMR 1H spectra, ITC, DNA polymerase stop assay, luciferase reporter assay and ChIP assay will evaluate the binding specificity in vitro and in vivo, comparing with c-myc, c-kit and telomeric G4-DNA. We will further determine the complex structures of RET G4-DNA with the small molecules specially targeting RET G4-DNA, optimize their chemical structures based on the results from the complex structures. Finally, we will further investigate whether the small molecules affect the transcription and expression of RET and downstream signal pathway, thus subsequently disrupting the initiation or progression of cancer cells. Hopefully, we will get a small molecule targeting RET G4-DNA specifically, and present molecular basis for rational drug design for RET-related cancer treatment.

原癌基因RET编码酪氨酸激酶受体，在乳腺癌等多种肿瘤中非正常高表达。其启动子含三个GC富集区，形成G-四联体（G4-DNA）结构，影响RET基因转录与表达。以RET启动子区的G4-DNA为靶点筛选小分子化合物配体，降低RET基因的表达，能克服传统酪氨酸激酶抑制剂药物的脱靶问题，为RET过表达类肿瘤提供候选药物分子。本项目利用分子模拟筛选RET G4-DNA结合的小分子化合物；以c-myc、c-kit启动子区和端粒G4-DNA为对照，利用NMR技术、CD、DSC、ITC、DNA聚合酶阻滞、荧光素酶报告基因和ChIP等实验，体内体外考察小分子与RET G4-DNA结合特异性；核磁结构解析确定小分子与RET G4-DNA复合物结构，优化小分子并测试其对乳腺癌细胞生长、迁移影响和对RET转录与信号通路的作用，得到特异性靶向RET G4-DNA的小分子，为合理化设计靶向RET抗肿瘤药物奠定基础。

RET是公认的原癌基因，在乳腺癌、胰腺癌等多种癌症中过表达，且其表达量与肿瘤大小和肿瘤恶性程度成正相关。研究报道RET基因启动子区域存在一段富含鸟嘌呤碱基的序列，在一价阳离子诱导下折叠形成G4-结构并对基因表达起调控的作用。本项目通过虚拟筛选结合核磁共振药物筛选的方法最终获得了天然产物秋水仙素可以特异性结合RET G4-DNA，而不与c-kit，c-myc 和 Tel26wt G4-DNA作用；秋水仙素体外稳定RET G4-DNA的结构，并在细胞内抑制RET mRNA的转录，影响肿瘤细胞的增殖。通过核磁共振技术，项目解析获得了RET G4-DNA与秋水仙素的复合物结构，以及广谱的G4-DNA配体黄连素与G4-DNA的结构，通过分析对比，我们发现 RET G4-DNA的loop区域碱基以及小分子化合物的立体构型对于结合特异性十分重要，为G4-DNA特异性化合物的筛选和优化提供参考。