Investigation of the design, structure and mechanism of Mena protein interaction inhibitors

项目摘要/摘要 肿瘤转移依赖于细胞骨架的协调过程，该过程由一种特征性的改变引起 特定运动性和肌动蛋白调节基因的表达。MENA，肌动蛋白Ena/Vasp家族成员 调节蛋白，在侵袭性癌细胞中高度上调。Ena/Vasp蛋白定位于肌动蛋白 通过其结构相似的EVH1结构域与其他蛋白质中的短线性基序(SLM)结合而组装。 MENA是侵袭性癌细胞特有的运动途径的组成部分。与入侵有关的 Mena的剪接变异体MenaINV比Mena对转移的影响要强得多，而且优先 在侵袭性癌细胞中表达。然而，对Mena和Mena的确切机械作用的确定 转移过程中的MenaINV已被证明具有挑战性。目前还没有分子上的解释 Mena及其类似物/异构体蛋白质相互作用性质的差异。设计 肽/迷你蛋白结合蛋白可以揭示结合特异性的分子决定因素，并激发/告知 铅抑制剂的设计。它们还可以用来探索蛋白质在细胞环境中的功能，以及 时间控制，提供治疗潜力的直接评估。这项提议的主要目标是 揭示MENA和MenaINV蛋白质相互作用特性差异的分子基础， 确定现有迷你蛋白抑制剂结合特异性的分子来源，并使用 这些信息用于设计和测试对偶数和异构体具有选择性、细胞通透性的迷你蛋白抑制剂 关于梅纳的。这一目标将通过应用一系列生物物理实验来实现，如核磁共振、SAXS、 X射线结晶学和结合分析揭示蛋白质相互作用差异的分子起源 在Mena和它的同源异构体之间。这些实验将揭示抑制剂的分子决定因素 结合的特异性，并有助于我们了解转移通过提供一个分子解释 Mena和MenaINV的区别。从这些实验中获得的生物物理信息将被 结合到使用尖端蛋白的平行对数和异构体特异性微型蛋白抑制剂的设计中 设计方法，包括基于结构的计算、重点文库设计和高通量筛选 技巧。除了各种新研究方法的培训外，这里概述的培训计划还包括 广泛开展科学交流，负责任地开展研究，建立科学网络， 教学、指导和管理技能。研究和培训将在博士的实验室进行。 艾米·基廷，一个处于蛋白质-蛋白质相互作用前沿的高度跨学科和协作的小组 和蛋白质设计领域。基廷实验室位于麻省理工学院生物系，是科赫实验室的一部分 综合癌症研究所，为培训、合作和研究提供理想的环境。

Project Summary/Abstract Cancer metastasis depends on coordinated cytoskeletal processes induced by a characteristic change in expression of specific motility and actin-regulatory genes. Mena, a member of the Ena/VASP family of actin regulatory proteins, is highly upregulated in invasive cancer cells. The Ena/VASP proteins localize to actin-based assemblies via their structurally similar EVH1 domains which bind to short linear motifs (SLMs) in other proteins. Mena is integral to motility pathways that are characteristic of invasive cancer cells. An invasion-associated splice variant of Mena, MenaINV, has far more potent effects on metastasis than Mena and is preferentially expressed in invasive cancer cells. However, determination of the precise mechanistic roles of Mena and MenaINV in metastatic processes has proven challenging. There is currently no molecular explanation for differences in the protein-protein interaction properties of Mena and its paralogs/isoforms. Designed peptide/mini-protein binders can reveal molecular determinants of binding specificity and inspire/inform the design of lead inhibitors. They can also be used to probe the function of proteins in their cellular context and with temporal control, providing a direct evaluation of therapeutic potential. The primary goal of this proposal is to uncover the molecular basis for differences in the protein-interaction properties of Mena and MenaINV, determine the molecular origin of the binding specificity of an existing mini-protein inhibitor, and use this information to design and test paralog- and isoform- selective, cell-permeable mini-protein inhibitors of Mena. This goal will be accomplished by applying an array of biophysical experiments such as NMR, SAXS, X-ray crystallography and binding assays to uncover the molecular origin of protein-interaction differences between Mena and its paralogs/isoforms. These experiments will reveal molecular determinants of inhibitor binding specificity and contribute to our understanding of metastasis by providing a molecular explanation for differences between Mena and MenaINV. The biophysical information gained from these experiments will then be incorporated into the design of paralog- and isoform- specific mini-protein inhibitors using cutting-edge protein design methods, including structure-based computation, focused library design, and high throughput screening techniques. In addition to training in a variety of new research methods, the training plan outlined here includes extensive development of scientific communication, responsible conduct of research, scientific networking, teaching, mentoring and management skills. The research and training will take place in the laboratory of Dr. Amy Keating, a highly interdisciplinary and collaborative group at the forefront of the protein-protein interaction and protein design fields. The Keating lab is embedded in the MIT Biology department and part of the Koch Institute for Integrative Cancer Research, providing an ideal environment for training, collaboration, and research.