HTS for Direct Targeting of the Transcription Factor MITF

产品说明：我们的Multi-PI研究团队的目标是使用Scripps Institutional Drug Discovery Library将这些HTS就绪检测方法应用于完整的HTS活动中，以开发、验证和优化MITF的小分子抑制剂。小眼症相关转录因子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，约20%的黑色素瘤含有高水平的MITF。用siRNA敲低MITF在这些MITF扩增的肿瘤中诱导细胞凋亡，但在许多缺乏MITF扩增的黑素瘤中也诱导细胞凋亡，突出了它作为黑素瘤的假定靶点。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，克服了这一障碍。我们测定了MITF核心区的脱辅基形式和DNA结合复合物的晶体结构，证明了MITF作为一种持久的二聚体存在以发挥功能。令人惊讶的是，我们发现MITF作为一个超动态二聚体存在，这是不同于其他一般的bHLH-Lz转录因子。二聚体界面上的突变对MITF与DNA的结合及其转录活性是有害的。这些研究揭示了一个独特的机会，抑制MITF通过直接化学扰动其同源二聚体。朝着这个方向，我们已经开发了一种高通量筛选（HTS）兼容的主要测定，具体措施在体外MITF的二聚化。LOPAC的屏幕显示出显著高的信号/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效能。我们的核心目标团队是使用Scripps Institutional Drug Discovery Library（SDDL）将这些HTS就绪检测方法实施到完整的HTS活动中，以开发、验证和优化MITF的小分子抑制剂。如目标1中所述，我们将将该测定方法应用于1，536孔板格式，筛选来自Scripps Institutional Drug Discovery Library（SDDL）的10，000种代表性化合物的试验组，并使用该筛选来验证整个筛选过程以及测量命中率。一旦达到HTS自动化标准，将通过分层方法筛选> 640，000种化合物的多样化SDDL库来进行"全甲板" 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试验来验证筛选命中，提高其效力和选择性，并在体外和体内评估其作用机制和功效。总的来说，我们的生物化学和基于细胞的测定，沿着针对一系列机制筛选分析先导化合物，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.