Thermodynamic and Allosteric Basis of Paradoxical Activation in V600E Mutant BRAF Cancers

项目总结 蛋白激酶BRAF中激活的V600E突变是人类癌症的一个公认的驱动因素。这个 目前针对V600E BRAF开发的BRAF抑制剂的有效性受到药理学的限制 在最初治疗几个月后不可避免地产生抗药性。已经发现，这些 抑制剂有效地抑制V600E单体的活性，但不抑制V600E BRAF二聚体，并且 取而代之的是通过一种称为矛盾激活(PA)的现象来提升它们的活性。因此，分子损伤 促进V600E BRAF二聚体的形成是导致抗性的主要机制，并突出 目前的药物无法抑制V600E BRAF二聚体，这是一个严重的临床问题。确切的结构 然而，导致PA和使V600E BRAF二聚体不受抑制的机制并不是很好 明白了。这项提案的目标是剖析V600E的自然变构调节机制 BRAF二聚体，并确定抑制剂如何调制这些结构网络，以便了解 PA的分子基础。中心假设是V600E BRAF二聚体本质上是不对称的，原因是 跨二聚体界面的变构偶联，而抑制剂影响这种偶联以驱动形成 催化活性二聚体，只被一个抑制剂分子占据。这项工作的基本原理是 了解BRAF二聚体变构和抑制剂诱导的变构的关系可以为未来提供信息 药物开发努力创造抑制剂，可以调整这些结构效应，以完全抑制 V600E BRAF二聚体。核心假设将通过两个具体目标进行检验：1)量化BRAF 在RAF抑制剂存在下的二聚亲和力建立PA的热力学因素，以及2。 测量BRAF二聚的变构机制，并确定它们对抑制剂结合的响应。 这些目标将通过使用Förster共振能量转移(FRET)分析来实现，该分析可以 在有抑制剂存在的情况下，定量BRAF的二聚化，以及双电子-电子共振(DER)以 直接测量BRAF激酶结构域对二聚化和 抑制剂结合。这项建议的预期结果将进一步加深对结构的理解 BRAF-抑制剂相互作用的机制及其如何导致PA。因此，这些知识将奠定基础 为合理设计下一代抑制剂和改进治疗策略，有效地抑制 V600E BRAF二聚体，克服阻力。

PROJECT SUMMARY The activating V600E mutation in the protein kinase BRAF is a well-established driver of human cancer. The effectiveness of current BRAF inhibitors developed to target V600E BRAF is limited by pharmacological resistance that unavoidably develops only months after initial treatment. It has been discovered that these inhibitors effectively suppress the activity of V600E monomers, but do not inhibit V600E BRAF dimers, and instead elevate their activity through a phenomenon called paradoxical activation (PA). Thus, molecular lesions promoting the formation of V600E BRAF dimers are primary mechanisms leading to resistance, and highlights the inability of current drugs to inhibit V600E BRAF dimers as a severe clinical problem. The exact structural mechanisms that cause PA and make V600E BRAF dimers impervious to inhibition, however, are not well understood. The goal of this proposal is to dissect the natural allosteric mechanisms of regulation of the V600E BRAF dimer, and determine how inhibitors modulate these structural networks in order to understand the molecular basis of PA. The central hypothesis is that The V600E BRAF dimer is intrinsically asymmetric due to allosteric coupling across the dimer interface, and inhibitors affect this coupling to drive the formation of catalytically active dimers that are only occupied by one inhibitor molecule. The rationale for this work is that understanding the relationship between BRAF dimer allostery and inhibitor-induced allostery can inform future drug development efforts in creating inhibitors that can tune these structural effects to completely inhibit the V600E BRAF dimer. The central hypothesis will be tested through two specific aims: 1.) Quantify BRAF dimerization affinity in the presence of RAF inhibitors to establish the thermodynamic factors of PA, and 2.) Measure the allosteric mechanisms of BRAF dimerization and determine their response to inhibitor binding. These aims will be achieved through the use of a Förster resonance energy transfer (FRET) assay that can quantify BRAF dimerization in the presence of inhibitors, and double electron-electron resonance (DEER) to directly measure conformational rearrangements of the BRAF kinase domain in response to dimerization and inhibitor binding. The expected results from this proposal will further the understanding of the structural mechanisms of BRAF-inhibitor interactions and how they lead to PA. This knowledge will thus lay the groundwork for the rational design of next-generation inhibitors and improved therapeutic strategies to effectively inhibit the V600E BRAF dimer and overcome resistance.