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

• 批准号: 10540319
• 负责人: Damien M Rasmussen
• 金额: $ 3.35万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/10540319
• 关键词: Address; Affect; Affinity; Aftercare; Allosteric Regulation; Automobile Driving; BRAF gene; Binding; Biochemical; Biological Assay; Clinical; Coupled; Coupling; Data; Development; Dimerization; Effectiveness; Electrons; Event; Fluorescence Resonance Energy Transfer; Future; Goals; Human; Knowledge; Lesion; Link; Malignant Neoplasms; Measures; Mediating; Metastatic Melanoma; Modeling; Molecular; Molecular Conformation; Mutation; Outcome; Pharmaceutical Preparations; Phosphotransferases; Protein Kinase; Proto-Oncogene Proteins B-raf; Protomer; Regulation; Resistance; Skin Cancer; Testing; Therapeutic; Thermodynamics; Transactivation; Work; biophysical techniques; dimer; drug development; improved; inhibitor; insight; monomer; mutant; next generation; novel; pharmacologic; rational design; resistance mechanism; response; success

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.

项目摘要 蛋白激酶BRAF中的激活V600E突变是人类癌症的公认驱动因素。的 目前开发的靶向V600E BRAF的BRAF抑制剂的有效性受到药理学的限制， 在最初治疗后几个月就产生了耐药性。已发现这些 抑制剂有效抑制V600E单体的活性，但不抑制V600E BRAF二聚体，和 而是通过一种称为反常激活（PA）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抗性的主要机制，并强调 目前的药物不能抑制V600E BRAF二聚体，这是一个严重的临床问题。准确的结构 然而，导致PA和使V600E BRAF二聚体不受抑制的机制并不清楚， 明白本提案的目的是剖析V600 E调节的天然变构机制。 BRAF二聚体，并确定抑制剂如何调节这些结构网络，以了解 PA的分子基础中心假设是V600E BRAF二聚体本质上是不对称的， 通过二聚体界面的变构偶联，和抑制剂影响这种偶联，以驱动形成 催化活性二聚体仅被一个抑制剂分子占据。这项工作的基本原理是， 了解BRAF二聚体变构与受体诱导的变构之间的关系， 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测试：1。定量BRAF 在RAF抑制剂存在下的二聚化亲和力，以建立PA的热力学因子，和2.） 测量BRAF二聚化的变构机制并确定其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）试验来实现，该试验可以 在抑制剂存在下定量BRAF二聚化，和双电子-电子共振（DEER）， 直接测量响应二聚化的BRAF激酶结构域的构象重排， 抑制剂结合。这一建议的预期结果将进一步了解结构 BRAF-抑制剂相互作用的机制以及它们如何导致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，以有效抑制 V600E BRAF二聚体和克服耐药。