HTS for Direct Targeting of the Transcription Factor MITF

描述：我们的多PI研究团队的目标是利用斯克里普斯机构药物发现库来开发、验证和优化MITF的小分子抑制剂，将这些HTS就绪的分析应用到全面的HTS活动中。小眼炎相关转录因子MITF是黑素细胞分化的主要调节因子，也是黑色素瘤的癌基因，约20%的黑色素瘤含有高水平的MITF。用siRNA敲除MITF可诱导这些MITF扩增的肿瘤中的细胞凋亡，但在许多缺乏MITF扩增的黑色素瘤中也能诱导细胞凋亡，这突出了它是黑色素瘤的假定靶点。然而，直接用药物抑制剂调节MITF仍然具有挑战性，主要是因为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核心区在脱氧核糖核酸和DNA结合复合体中的晶体结构，证明了MITF以持久性二聚体的形式存在。令人惊讶的是，我们发现MITF以一种不同于其他普通bHLH-LZ转录因子的高动态二聚体的形式存在。二聚体界面的突变对MITF与DNA的结合及其转录活性是有害的。这些研究揭示了通过对MITF同源二聚体的直接化学干扰来抑制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)兼容的初级检测方法，专门测量MITF在体外的二聚化。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，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，它们的细胞渗透衍生物可以抑制MITF转录活性、原代黑素细胞色素形成和黑色素瘤细胞生长，并具有μM效力。 我们的中心目标团队是利用斯克里普斯机构药物发现库(SDDL)将这些HTS就绪分析应用到全面的HTS活动中，以开发、验证和优化MITF的小分子抑制剂。如目标1所述，我们将把这项检测小型化到1,536孔板格式，从斯克里普斯机构药物发现库(SDDL)中筛选10,000种具有代表性的化合物，并使用这一筛选来验证整体筛选过程以及测量命中率。一旦达到HTS自动化标准，将通过分级方法对640,000种化合物的多样化SDDL库进行筛选，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分析来验证筛选命中，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效果。总体而言，我们的生化和基于细胞的分析，以及针对一系列机械筛查的先导化合物的分析，将推动MITF新型选择性分子调节剂的发现，用于癌症治疗。

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 μM 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.