Allosteric inhibitors targeting oncogenic BRAF V600E dimers

MAPK信号通路的激活增加可诱发恶性肿瘤。人胆囊癌基因突变V600E 在几乎50%的黑色素瘤肿瘤中都发现了BRAF。此外，相当大比例的BRAFV600E 在结直肠癌和甲状腺癌中分别发现了约10%和40%的突变。FDA批准的皇家空军 黑色素瘤抑制剂在BRAFV600E患者中显示出显著的反应，然而，治疗几乎领先于 不可避免地会产生后天抵抗力。同样，携带BRAFV600E的结直肠癌和甲状腺癌在很大程度上 对RAF抑制剂治疗耐药。多项研究表明，内源性或抑制物诱导的 BRAFV600E二聚化在抗性机制中起关键作用。在黑色素瘤中，BRAFV600E通常 作为活性单体，然而，在肿瘤活性剪接BRAFV600E二聚体的重要子集中 调解原发抗性。同样，在很大一部分RAS激活的上游癌症中，如结直肠癌和 甲状腺、BRAFV600E二聚体升高，上述药物无效。根据临床相关性 BRAFV600E二聚体在几种肿瘤中驱动耐药性，我们目前迫切需要开发 针对有效和特定活性的BRAF二聚体的新型抑制剂。我们最近演示了 RAF抑制剂的结构和生化效应与其临床表现的相关性。使用这个 在知识方面，我们开始了一项系统的探索，寻找能够识别BRAFV600E二聚体的新型抑制剂。 我们发现FDA批准的药物Ponatinib就是这样一种抑制剂。在广泛的初步研究中，我们 表征了波纳替尼对黑色素瘤、结直肠癌等癌症的作用，并获得了其共晶体 采用BRAFV600E和BRAFWT的结构。值得注意的是，BRAF/Ponatinib结构显示出 完美对称的BRAF二聚体和变构抑制剂结合模式，在任何BRAF中都是前所未有的 到目前为止的抑制剂。我们的观察为下一步的药物设计创造了一个特殊的机会- 产生变构BRAF抑制剂，专门抑制BRAF二聚体。基于这些观察，我们 合成了一种波纳替尼杂化化合物，测定了它的共晶结构，并验证了它的生化和 细胞活动。我们的结果显示，与BRAFV600E二聚体相比，BRAFV600E二聚体具有优异的效力和特异性 为此类一流变构BRAF的开发提供了坚实的基础 抑制剂。在这项提案中，我们将1)设计和合成与小说结合的波纳替尼杂化化合物 BRAF的变构口袋，并显示出与BRAFV600E二聚体更好的结合和特异性 ADME特性，2)强有力地验证和优化变构BRAF抑制剂的效力和特异性 生化、生物物理和细胞实验；3)研究其作用的细胞机制。 改进的变构BRAFV600E抑制剂及其在小鼠肿瘤模型中的治疗潜力。这个项目 将调查一个广泛未得到满足的治疗机会，即BRAFV600E的药理靶向- 二聚化依赖的肿瘤，对当前的治疗方法耐药。

Increased activation of the MAPK signaling pathway can induce malignancies. Oncogenic mutation V600E in BRAF is found in almost 50% of melanoma tumors. In addition, substantial percentages of BRAFV600E mutants are found in colorectal and thyroid cancers, about 10% and 40%, respectively. FDA-approved RAF inhibitors for melanoma show remarkable responses in BRAFV600E patients, however, treatment leads almost invariably to acquired resistance. Similarly, colorectal and thyroid cancers with BRAFV600E are largely resistant to RAF inhibitor treatment. Several studies have demonstrated that intrinsic or inhibitor-induced BRAFV600E dimerization plays a key role in the resistance mechanism. In melanoma, BRAFV600E usually acts as an active monomer, however, in a significant subset of tumors active spliced BRAFV600E dimers mediate primary resistance. Likewise, in large portions of upstream RAS-activated cancers like colorectal and thyroid, BRAFV600E dimers are promoted and the above drugs are ineffective. In light of the clinical relevance of BRAFV600E dimers to drive resistance in several tumors, we are currently in pressing need to develop novel inhibitors that target potently and specifically active BRAF dimers. We recently demonstrated the correlation of structural and biochemical effects of RAF inhibitors with their clinical manifestations. Using this knowledge, we initiated a systematic quest for novel inhibitors that specifically recognize BRAFV600E dimers. We discovered that Ponatinib, an FDA-approved drug, is such an inhibitor. In extensive preliminary studies, we characterized the effect of Ponatinib in melanoma, colorectal and other cancers and obtained its co-crystal structure with BRAFV600E and BRAFWT. Remarkably, the BRAF/Ponatinib structures demonstrated a perfectly symmetrical BRAF dimer and an allosteric inhibitor-binding mode, unprecedented for any BRAF inhibitor to date. Our observations generate an exceptional opportunity for drug design towards next- generation allosteric BRAF inhibitors that specifically inhibit BRAF dimers. Based on these observations, we created a ponatinib-hybrid compound, determined its co-crystal structure and validated its biochemical and cellular activity. Our results demonstrate excellent potency and specificity for BRAFV600E dimers compared to BRAFV600E monomers and provide a solid basis for the development of such first-in-class allosteric BRAF inhibitors. In this proposal, we will 1) design and synthesize ponatinib-hybrid compounds that bind to the novel allosteric pocket of BRAF and display improved binding and specificity to BRAFV600E dimers and desirable ADME properties, 2) robustly validate and optimize the potency and specificity of allosteric BRAF inhibitors in biochemical, biophysical and cellular experiments and 3) investigate the cellular mechanism of action of improved allosteric BRAFV600E inhibitors and their therapeutic potential in mouse tumor models. This project will investigate a broadly unmet therapeutic opportunity, namely the pharmacological targeting of BRAFV600E- dimerization dependent tumors that are resistant to current treatments.