Allosteric inhibitors targeting oncogenic BRAF V600E dimers

针对致癌 BRAF V600E 二聚体的变构抑制剂

• 批准号: 10520017
• 负责人: Evripidis Gavathiotis
• 金额: $ 44.2万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10520017
• 关键词: Address; BRAF gene; Binding; Biochemical; Biological Assay; Biophysics; Cell Line; Cells; Clinical; Colorectal; Colorectal Cancer; Colorectal Neoplasms; Computing Methodologies; Development; Dimerization; Drug Design; Drug Kinetics; Drug resistance; FDA approved; Feedback; Generations; Goals; Guanosine Triphosphate; Homo; Hybrids; In Vitro; Investigation; Knowledge; Lead; Light; MAP Kinase Gene; MAPK Signaling Pathway Pathway; MEKs; Malignant Neoplasms; Malignant neoplasm of thyroid; Mediating; Medical; Melanoma Cell; Modeling; Mus; Mutate; Mutation; Oncogenic; PTPN11 gene; Pathway interactions; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacodynamics; Play; Process; Prognosis; Progression-Free Survivals; Property; Quantitative Structure-Activity Relationship; RNA Splicing; Regulation; Resistance; Series; Signal Transduction; Solid; Specificity; Structure; Therapeutic; Therapeutic Index; Thyroid Gland; Toxic effect; Translating; Validation; Variant; Xenograft Model; cancer cell; clinically relevant; computational pipelines; design; dimer; drug development; drug discovery; experimental study; improved; in silico; in vivo; inhibitor; innovation; insight; lead optimization; melanoma; monomer; multidisciplinary; mutant; neoplastic cell; next generation; novel; pharmacologic; prototype; resistance mechanism; response; screening; thyroid neoplasm; tumor

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.

MAPK信号通路的激活增加可诱导恶性肿瘤。肿瘤基因突变V600 E BRAF存在于几乎50%的黑色素瘤中。此外，BRAFV 600 E的显著百分比 在结直肠癌和甲状腺癌中发现了突变体，分别约为10%和40%。FDA批准的RAF 黑色素瘤抑制剂在BRAFV 600 E患者中显示出显著的反应，然而，治疗几乎 不可避免地会产生后天抵抗力。类似地，BRAFV 600 E的结直肠癌和甲状腺癌在很大程度上是 对RAF抑制剂治疗有抗性。一些研究表明，内在的或神经诱导的 BRAFV 600 E二聚化在耐药机制中起关键作用。在黑色素瘤中，BRAFV 600 E通常 然而，在肿瘤活性剪接BRAFV 600 E二聚体的重要亚组中， 介导主要抗性。同样，在大部分上游RAS激活的癌症如结肠直肠癌和结肠癌中， 甲状腺，BRAFV 600 E二聚体被促进，并且上述药物无效。根据临床相关性 BRAFV 600 E二聚体在几种肿瘤中驱动耐药性，我们目前迫切需要开发 有效和特异性靶向活性BRAF二聚体的新型抑制剂。我们最近展示了 RAF抑制剂的结构和生化作用与其临床表现的相关性。使用此 基于这些知识，我们开始了对特异性识别BRAFV 600 E二聚体的新型抑制剂的系统性探索。 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 表征了泊那替尼在黑色素瘤、结直肠癌和其他癌症中的作用，并获得了其共晶 采用BRAFV 600 E和BRAFWT结构。值得注意的是，BRAF/泊那替尼结构证明了 完全对称的BRAF二聚体和变构的底物结合模式，对于任何BRAF都是前所未有的 迄今为止的抑制剂。我们的观察为药物设计提供了一个特殊的机会， 产生特异性抑制BRAF二聚体的变构BRAF抑制剂。根据这些观察，我们 创造了一种泊那替尼混合化合物，确定了它的共晶体结构，并验证了它的生物化学和 细胞活动我们的结果表明，与对照组相比，BRAFV 600 E二聚体具有优异的效力和特异性。 BRAFV 600 E单体的合成，为开发此类一流的变构BRAF提供了坚实的基础 抑制剂的在这项提案中，我们将1）设计和合成与新的 在一些实施方案中，所述组合物是BRAF的变构口袋，并显示出对BRAFV 600 E二聚体的改善的结合和特异性， ADME特性，2）稳健地验证和优化变构BRAF抑制剂在抗肿瘤治疗中的效力和特异性。 生物化学、生物物理学和细胞实验，以及3）研究细胞作用机制， 改进的变构BRAFV 600 E抑制剂及其在小鼠肿瘤模型中的治疗潜力。这个项目 将研究一个广泛未满足的治疗机会，即BRAFV 600 E的药理学靶向- 二聚化依赖性肿瘤对目前的治疗具有抗性。