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

V600E 突变 BRAF 癌症中矛盾激活的热力学和变构基础

基本信息

批准号：
10323651
负责人：
Damien M Rasmussen
金额：
$ 3.25万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-04 至 2024-01-03
项目状态：
已结题

项目摘要

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.

项目概要蛋白激酶 BRAF 中的激活 V600E 突变是公认的人类癌症驱动因素。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初步治疗后仅几个月就不可避免地出现耐药性。抑制剂有效抑制V600E单体的活性，但不抑制V600E BRAF二聚体，并且相反，通过一种称为矛盾激活（PA）的现象来提高它们的活性，因此，分子损伤。促进 V600E BRAF 二聚体的形成是导致耐药性的主要机制，并强调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。然而，导致 PA 并使 V600E BRAF 二聚体不受抑制的机制尚不明确该提案的目标是剖析 V600E 调节的自然变构机制。 BRAF 二聚体，并确定抑制剂如何调节这些结构网络，以了解 PA 的分子基础是 V600E BRAF 二聚体本质上是不对称的。跨二聚体界面的变构偶联，抑制剂影响这种偶联以驱动形成仅被一个抑制剂分子占据的催化活性二聚体。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之间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。中心假设将通过两个具体目标进行检验：1.) 量化 BRAF RAF 抑制剂存在下的二聚亲和力以确定 PA 的热力学因素，2.) 测量 BRAF 二聚化的变构机制并确定其对抑制剂结合的反应。这些目标将通过使用福斯特共振能量转移（FRET）检测来实现，该检测可以在存在抑制剂的情况下量化 BRAF 二聚化，并使用双电子-电子共振 (DEER) 来直接测量 BRAF 激酶结构域响应二聚化的构象重排该提案的预期结果将进一步加深对结构的理解。 BRAF 与抑制剂相互作用的机制以及它们如何导致 PA 奠定了基础。合理设计下一代抑制剂并改进治疗策略以有效抑制 V600E BRAF 二聚体并克服阻力。