HTS for Direct Targeting of the Transcription Factor MITF

描述：我们的Multi-PI研究团队的目标是利用Scripps机构药物发现库开发、验证和优化MITF的小分子抑制剂，将这些HTS-ready检测方法应用到一个完整的HTS-活动中。小眼相关转录因子MITF是黑素细胞分化的主要调节因子，也是黑色素瘤的致癌基因，大约20%的黑色素瘤含有高水平的MITF。在这些MITF扩增的肿瘤中，用siRNA敲低MITF可诱导细胞凋亡，但在许多缺乏MITF扩增的黑色素瘤中也可诱导细胞凋亡，这表明MITF是黑色素瘤的一个假定靶点。然而，用药物抑制剂直接调节MITF仍然具有挑战性，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，克服了这个障碍。我们在载脂蛋白和dna结合复合体中测定了MITF核心区的晶体结构，证明了MITF作为一个持续的二聚体存在并发挥作用。令人惊讶的是，我们发现MITF作为一种超动态二聚体存在，与其他一般bHLH-Lz转录因子不同。二聚体界面上的突变对MITF与DNA的结合及其转录活性有害。这些研究揭示了一个独特的机会，抑制MITF的直接化学扰动其同型二聚体。朝着这个方向，我们开发了一种高通量筛选（HTS）兼容的初级试验，专门测量体外MITF的二聚化。LOPAC的屏幕显示出显着的高信号/背景窗口，再现性和Z'为0.81。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特异性干扰物，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黑色素瘤细胞的生长。M效力。我们的中心目标团队是利用Scripps机构药物发现库（SDDL）开发、验证和优化MITF的小分子抑制剂，将这些hts就绪的检测方法应用到一个完整的hts活动中。正如Aim 1中概述的那样，我们将把该试验缩小到1536孔板格式，筛选来自Scripps机构药物发现库（SDDL）的10,000个代表性化合物的试点集，并使用该筛选来验证整个筛选过程以及测量成功率。一旦达到HTS自动化标准，将开展“全层”HTS活动，通过分层方法筛选bbbb640,000个化合物的各种SDDL库，以发现Aim 2中的化合物。在Aim 3中，我们将使用一系列后续试验来验证筛选命中，提高它们的效力和选择性，并评估它们在体外和体内的作用机制和功效。总的来说，我们的生化和基于细胞的分析，以及针对一系列机制筛选的分析先导化合物，将推动发现用于癌症治疗的MITF新型和选择性分子调节剂。

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.