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

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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.