HTS for Direct Targeting of the Transcription Factor MITF

用于直接靶向转录因子 MITF 的 HTS

基本信息

批准号：
8762056
负责人：
Min Guo
金额：
$ 48.27万
依托单位：
SCRIPPS FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8762056
关键词：
Apoptosis Automation Binding Biochemical Biological Assay Biosensor Cell Line Cells Chemicals Collection Complex DNA DNA Binding Dimerization Florida Fluorescein-5-isothiocyanate Fluorescence Anisotropy Gene Targeting Genetic Transcription Goals Homodimerization Human In Vitro Kinetics Lead Libraries MAPK14 gene Measurement Measures Melanoma Cell Miniaturization Modeling Molecular Mutation Oncogenes Pharmaceutical Preparations Phase Pigments Process Regimen Reporter Genes Reproducibility Research Reverse Transcriptase Polymerase Chain Reaction Screening Result Series Signal Transduction Small Interfering RNA Specificity Structure TFE3 gene Testing Time Transgenic Organisms Validation Zebrafish base cancer therapy cell growth cheminformatics counterscreen dimer drug discovery follow-up high throughput screening improved in vivo inhibitor/antagonist meetings melanocyte melanoma microphthalmia-associated transcription factor miniaturize neoplastic cell novel novel therapeutics pharmacophore public health relevance scaffold screening small molecule small molecule libraries transcription factor tumor tumorigenesis

项目摘要

DESCRIPTION: The goals of our Multi-PI research team are to implement these HTS-ready assays into a full HTS- campaign using the Scripps Institutional Drug Discovery Library to develop, validate and optimize small molecule inhibitors of MITF. Microphthalmia-associated transcription factor MITF is a master regulator of melanocyte differentiation and an oncogene in melanoma, with approximately 20% of melanomas containing high-level MITF. Knockdown of MITF with siRNA induces apoptosis in these MITF-amplified tumors, but also in many melanomas lacking MITF amplification, highlighting it as a putative target for melanoma. However, directly modulating MITF with pharmacologic inhibitors remains challenging, mainly due to the lack of a defined mechanism for inhibition. Our recent research has made breakthroughs that overcame this obstacle. We determined crystal structures of the MITF core region in the apo-form and in the DNA-binding complex, demonstrated that MITF exists as a persistent dimer to function. Surprisingly, we discovered that MITF exists as a hyper-dynamic dimer that is distinct from other general bHLH-Lz transcription factors. Mutations on the dimer interface are deleterious for MITF binding to DNA and its transcriptional activity. These studies revealed a unique opportunity for inhibiting MITF by a direct chemical perturbation of its homodimer. Towards this direction, we have developed a high-throughput screening (HTS)-compatible primary assay that specifically measures the dimerization of MITF in vitro. A screen of the LOPAC has shown a significantly high signal/background window, reproducibility, and a Z' of 0.81. Importantly, the proof-of-principle studies using 3K trial screening have yielded multiple MITF specific disruptors and, their cell permeable derivatives could inhibit MITF transcription activity, pigment formation of primary melanocyte cells and melanoma cell growth with ?M potency. Our central goal team are to implement these HTS-ready assays into a full HTS-campaign using the Scripps Institutional Drug Discovery Library (SDDL) to develop, validate and optimize small molecule inhibitors of MITF. As outlined in Aim 1, we will miniaturize this assay to the 1,536-well plate format, screen a pilot set of 10,000 representative compounds from the Scripps Institutional Drug Discovery Library (SDDL), and use this screen to validate the overall screening process as well as gauge hit rate. Once the HTS automation criteria are met, a "full-deck" HTS-campaign will be carried out by screening the diverse SDDL library of >640,000 compounds in a tiered approach to discover compounds in Aim 2. In Aim 3, we will use a cascade of follow-up assays to validate screening hits, improve their potency and selectivity, and evaluate their mechanism of action and efficacy both in vitro and in vivo. Collectively, our biochemical and cell-based assays, along with profiling lead compounds against a series of mechanistic screens will drive the discovery of MITF novel and selective molecular modulators for cancer treatment.

描述：我们的多PI研究团队的目标是使用Scripps机构药物发现库将这些HTS就绪的测定方法实施到完整的HTS运动中，以开发，验证和优化MITF的小分子抑制剂。与肉毒杆菌相关的转录因子MITF是黑色素细胞分化的主要调节剂和黑色素瘤中的癌基因，约有20％的黑色素瘤含有高级MITF。用siRNA敲除MITF会诱导这些MITF放大肿瘤的凋亡，但在许多缺乏MITF扩增的黑色素瘤中也诱导了凋亡，这突出了它是黑色素瘤的推定靶标。但是，用药理学抑制剂直接调节MITF仍然具有挑战性，这主要是由于缺乏确定的抑制机制。我们最近的研究取得了突破，克服了这一障碍。我们确定了Apo形式中MITF核心区域的晶体结构和DNA结合复合物中的晶体结构，证明MITF作为持续的功能二聚体存在。令人惊讶的是，我们发现MITF是一种与其他一般BHLH-LZ转录因子不同的超动态二聚体。二聚体界面上的突变对于MITF与DNA及其转录活性的结合是有害的。这些研究揭示了通过其同型二聚体的直接化学扰动抑制MITF的独特机会。朝这个方向朝着这个方向开发了高通量筛选（HTS）兼容的主要测定法，该测定法专门测量了MITF体外的二聚化。 LOPAC的屏幕显示出明显高的信号/背景窗口，可重复性，Z'为0.81。重要的是，使用3K试验筛选的原理研究产生了多个MITF特异性破坏者，并且它们的细胞可渗透衍生物可以抑制MITF转录活性，原代黑素细胞细胞的色素形成和黑色素瘤细胞的生长。我们的中心目标团队是使用Scripps机构药物发现库（SDDL）将这些可HTS就绪的测定方法实施到完整的HTS-Campaign中，以开发，验证和优化MITF的小分子抑制剂。如AIM 1中概述的那样，我们将将该测定法对1,536孔板格式进行小型化，筛选了Scripps机构药物发现文库（SDDL）的10,000种代表性化合物的试点，并使用此筛查来验证整体筛选过程以及总计命中率。一旦满足了HTS自动化标准，将通过在AIM 2中筛选> 640,000种化合物的各种SDDL库来进行“全面” HTS-Campaign。体内。总体而言，我们的生化和基于细胞的测定法以及针对一系列机械筛选的铅化合物分析将推动MITF小说和选择性分子调节剂的发现进行癌症治疗。