微小眼转录因子（MITF）调节黑素细胞的发育，是黑色素瘤"特异性存活"癌基因。MITF对黑色素瘤的发生、进展和复发至关重要，一直被认为是一个重要的治疗靶点；然而，由于缺乏药物设计的配体结合口袋直接抑制MITF是不可能的。在这里，我们的结构分析表明，MITF的结构是超动态的，因为它有一个3个残基插入的反向亮氨酸拉链。这个动态MITF对二聚体破裂突变非常脆弱，因为我们发现在人类Waardenburg综合症2A发生MITF功能丧失突变时，这些突变通常位于二聚体接口，并相应破坏了二聚体形成能力。这些观察结果表明，使用能够破坏MITF二聚体的小分子有独特的机会抑制MITF。在对654,650个化合物进行高通量筛选后，我们发现化合物TT-012可以特异性地结合动态MITF，并破坏后者的二聚体形成和DNA结合能力。使用染色质免疫沉淀分析和RNA测序，我们展示了TT-012抑制B16F10黑色素瘤细胞中MITF的转录活性。此外，TT-012抑制高MITF黑色素瘤细胞的生长，并能在动物模型中抑制肿瘤的生长和转移，对肝脏和免疫细胞的耐受性较高。总之，本研究展示了一种独特的黑色素瘤癌基因MITF的超动态二聚体接口，并揭示了一种新的抑制MITF活性的治疗方法。 © 2022年。版权所有者授权中国科学院细胞生物学重点实验室。

Microphthalmia transcription factor (MITF) regulates melanocyte development and is the "lineage-specific survival" oncogene of melanoma. MITF is essential for melanoma initiation, progression, and relapse and has been considered an important therapeutic target; however, direct inhibition of MITF through small molecules is considered impossible, due to the absence of a ligand-binding pocket for drug design. Here, our structural analyses show that the structure of MITF is hyperdynamic because of its out-of-register leucine zipper with a 3-residue insertion. The dynamic MITF is highly vulnerable to dimer-disrupting mutations, as we observed that MITF loss-of-function mutations in human Waardenburg syndrome type 2 A are frequently located on the dimer interface and disrupt the dimer forming ability accordingly. These observations suggest a unique opportunity to inhibit MITF with small molecules capable of disrupting the MITF dimer. From a high throughput screening against 654,650 compounds, we discovered compound TT-012, which specifically binds to dynamic MITF and destroys the latter's dimer formation and DNA-binding ability. Using chromatin immunoprecipitation assay and RNA sequencing, we showed that TT-012 inhibits the transcriptional activity of MITF in B16F10 melanoma cells. In addition, TT-012 inhibits the growth of high-MITF melanoma cells, and inhibits the tumor growth and metastasis with tolerable toxicity to liver and immune cells in animal models. Together, this study demonstrates a unique hyperdynamic dimer interface in melanoma oncoprotein MITF, and reveals a novel approach to therapeutically suppress MITF activity.© 2022. The Author(s) under exclusive licence to Center for Excellence in Molecular Cell Science, CAS.