Project 3: Protein Design for Selective Interference with LPA Signaling in Colon Cancer

项目 3：选择性干扰结肠癌 LPA 信号传导的蛋白质设计

• 批准号: 8813298
• 负责人: Gevorg Grigoryan
• 金额: $ 27.42万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8813298
• 关键词: Address; Affinity; American; Attenuated; Binding; Biological Assay; Biomedical Research; Cause of Death; Cell physiology; Cells; Cessation of life; Colon Carcinoma; Colorectal Cancer; Complex; Computing Methodologies; Data; Development; Disease; Disease Pathway; Edg4 Protein; Ensure; Environment; Event; Family; Fluorescence; Funding; Goals; Health; In Vitro; Institutes; Life; Link; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mentors; Mitogens; Neoplasm Metastasis; Oncogenic; Outcome; Patients; Peptides; Positioning Attribute; Protein Engineering; Protein Family; Proteins; Proteomics; Publishing; Reagent; Recruitment Activity; Relapse; Research; Scaffolding Protein; Signal Pathway; Signal Transduction; Specificity; Techniques; Technology; Tertiary Protein Structure; Testing; Therapeutic; United States; Validation; Work; base; biophysical properties; cancer cell; career; colon tumorigenesis; design; functional outcomes; inhibitor/antagonist; lysophosphatidic acid; member; migration; novel; protein purification; research and development; research study; sodium-hydrogen exchanger regulatory factor; targeted agent; therapeutic development; therapeutic target; tool

Colorectal cancer was the third-most common cause of deaths from cancer in the United States in 2013. This disease claims over 50,000 lives and afflicts over 140,000 people annually in the US, underscoring a clear and urgent need for better control of this incurable malignancy. The goal of this study is to attenuate oncogenic activities in colon cancer cells by inhibiting a key proliferatory signaling pathway mediated by the powerful mitogen lysophosphatidic acid (LPA). In colon cancer, LPA signals through the LPA2 receptor, which recruits to its C-terminus a scaffolding protein NHERF-2. This recruitment occurs though a PDZ domain of NHERF-2 (N2P2)-a ubiquitous protein module that recognizes C-termini of partner proteins. Our goal is to inhibit the NHERF-2:LPA2 complex, and with it LPA signaling, using designed peptides that associate tightly with N2P2. Complicating this approach is the recent discovery that another PDZ-containing protein, MAGI-3, competes with NHERF-2 for binding to LPA2 and alters the functional outcome. Binding of NHERF-2 to LPA2 increases oncogenic signaling, while binding of MAGI-3 (via its PDZ domain M3P6) decreases it, underscoring the need for selectivity in N2P2 targeting. We are uniquely capable of providing such selectivity using our computational technologies that enable us to target a single member in a family of domains. Thus, our central hypothesis is that peptides designed to inhibit N2P2 will down-regulate oncogenic LPA signaling and that our computational methodology will uncover efficacious inhibitors with high N2P2 affinity and excellent selectivity against M3P6 and other PDZ domains, in vitro and in cells. The study will test our hypothesis through three specific aims. Aim 1 will establish that peptides already designed to target N2P2 and not M3P6 down-regulate LPA signaling in colon cancer cells. Aim 2 will test the hypothesis that our computational technology can produce peptides selective for N2P2 across the PDZome by designing and biophysically characterizing such peptides. Finally, Aim 3 will use a proteomic/mass-spectrometry assay along with fluorescence correlation techniques to quantify the selectivity of our peptides within cells and link biophysical parameters with function. We expect to 1) validate N2P2 targeting as a viable strategy for reducing LPA oncogenicity, 2) establish the functional relevance of selectivity against M3P6 (Aim 1) and other PDZ domains (Aims 2, 3), and 3) produce reagents suitable to initiate the development of colon cancer therapeutics. Our goals fit well with the overall theme of iTarget centered on biomolecular targeting. Being a part of the iTarget will accelerate our work significantly. Interactions with Drs. Kettenbach and McLellan will provide critical guidance in our proteomic/MS experiments and structural characterizations, respectively. Strong mentoring by Drs. Madden and Gerber on career and research matters will ensure high competitiveness for external funding. VMIC and MTC cores will be invaluable for in-cell and biophysical experiments (VMIC) and for expression and purification of proteins (MTC). Finally, iTarget will provide for a stimulating intellectual environment in which our research will thrive.

结直肠癌是2013年美国癌症死亡的第三大常见原因。这 在美国，这种疾病每年夺去5万多人的生命，折磨着14万多人，这突出了一个明确的， 迫切需要更好地控制这种无法治愈的恶性肿瘤。本研究的目的是减少致癌性 活性在结肠癌细胞通过抑制一个关键的增殖信号通路介导的强大的 丝裂原溶血磷脂酸（LPA）。在结肠癌中，LPA通过LPA 2受体发出信号，LPA 2受体招募， 其C-末端是支架蛋白NHERF-2。这种募集通过NHERF-2的PDZ结构域发生 （N2 P2）-一种普遍存在的蛋白质模块，识别伴侣蛋白的C-末端。我们的目标是抑制 NHERF-2：LPA 2复合物，以及与其LPA信号传导，使用与N2 P2紧密结合的设计肽。 使这种方法复杂化的是，最近发现另一种含有PDZ的蛋白质MAGI-3与PDZ竞争。 与NHERF-2结合LPA 2并改变功能结果。NHERF-2与LPA 2的结合增加 致癌信号传导，而MAGI-3的结合（通过其PDZ结构域M3 P6）降低了它，强调了需要 用于N2 P2靶向的选择性。我们是唯一能够提供这种选择性使用我们的计算 这些技术使我们能够针对域家族中的单个成员。因此，我们的中心假设是 设计用于抑制N2 P2的肽将下调致癌LPA信号传导， 该方法将揭示具有高N2 P2亲和力和对M3 P6的优异选择性的有效抑制剂 和其他PDZ结构域。这项研究将通过三个具体目标来检验我们的假设。 目的1将确定已经设计为靶向N2 P2而非M3 P6的肽下调LPA信号传导 在结肠癌细胞中。目标2将测试我们的计算技术可以产生肽的假设 通过设计和生物药理学表征这样的肽，在整个PDZome中对N2 P2具有选择性。最后， 目标3将使用蛋白质组学/质谱分析沿着荧光相关技术来定量 我们的肽在细胞内的选择性，并将生物物理参数与功能联系起来。我们希望1） 验证N2 P2靶向作为降低LPA致癌性的可行策略，2）建立功能性 针对M3 P6（目标1）和其它PDZ结构域（目标2、3）的选择性的相关性，和3）产生试剂 适合于启动结肠癌治疗剂的开发。我们的目标与总的主题非常吻合， iTarget以生物分子靶向为中心。成为iTarget的一部分将大大加快我们的工作。 与凯滕巴赫博士和麦克莱伦博士的互动将为我们的蛋白质组学/MS实验提供重要指导 和结构特征。Madden博士和Gerber博士在职业生涯和 研究事项将确保对外部资金的高度竞争力。VMIC和MTC核心将是非常宝贵的 用于细胞内和生物物理实验（VMIC）以及蛋白质的表达和纯化（MTC）。最后， iTarget将为我们的研究提供一个令人兴奋的智力环境。