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斯克里普斯机构药物发现库将这些 HTS 就绪测定法实施到完整的 HTS 活动中，以开发、验证和优化 MITF 的小分子抑制剂。小眼相关转录因子 MITF 是黑色素细胞分化的主要调节因子，也是黑色素瘤中的癌基因，大约 20% 的黑色素瘤含有高水平的 MITF。用 siRNA 敲低 MITF 会诱导这些 MITF 扩增肿瘤的细胞凋亡，而且还会诱导许多缺乏 MITF 扩增的黑色素瘤发生细胞凋亡，这凸显了它是黑色素瘤的假定靶标。然而，用药物抑制剂直接调节 MITF 仍然具有挑战性，这主要是由于缺乏明确的抑制机制。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 形式和 DNA 结合复合物中 MITF 核心区域的晶体结构，证明 MITF 作为持久二聚体存在并发挥作用。令人惊讶的是，我们发现 MITF 作为超动态二聚体存在，与其他一般 bHLH-Lz 转录因子不同。二聚体界面上的突变对于 MITF 与 DNA 的结合及其转录活性是有害的。这些研究揭示了通过直接化学扰动同型二聚体来抑制 MITF 的独特机会。朝着这个方向，我们开发了一种与高通量筛选（HTS）兼容的初级测定，专门测量 MITF 的体外二聚化。 LOPAC 的屏幕显示出显着高的信号/背景窗口、再现性和 0.81 的 Z'。重要的是，使用 3K 试验筛选的原理验证研究已经产生了多种 MITF 特异性干扰剂，并且它们的细胞渗透性衍生物可以抑制 MITF 转录活性、原代黑素细胞的色素形成和具有 ?M 效力的黑素瘤细胞生长。我们团队的中心目标是使用斯克里普斯机构药物发现库 (SDDL) 将这些 HTS 就绪检测实施到完整的 HTS 活动中，以开发、验证和优化 MITF 小分子抑制剂。如目标 1 中所述，我们将将此测定小型化为 1,536 孔板格式，从斯克里普斯机构药物发现库 (SDDL) 筛选一组 10,000 种代表性化合物，并使用此筛选来验证整个筛选过程作为仪表命中率。一旦满足 HTS 自动化标准，将通过分层方法筛选超过 640,000 种化合物的多样化 SDDL 库来开展“全方位”HTS 活动，以发现目标 2 中的化合物。在目标 3 中，我们将使用一系列后续测定，以验证筛选命中，提高其效力和选择性，并评估其体外和体内的作用机制和功效。总的来说，我们的生化和细胞检测，以及针对一系列机制筛选的先导化合物分析，将推动 MITF 新型选择性分子调节剂的发现，用于癌症治疗。