UNS: Bi-cyclic peptides for specific inhibition of intracellular protein-protein interactions

1510845 Rao，Balaji M.细胞过程，如生长、死亡和分化，由细胞内蛋白质之间的相互作用调节。蛋白质-蛋白质相互作用的中断(PPI)是研究其在特定细胞环境中的意义的有用工具。该项目的目标是开发一类专门抑制感兴趣的PPI的化合物。具体地说，将开发针对与癌症和人类胚胎干细胞(HESCs)相关的模型PPI的抑制剂化合物。如果成功，该项目的结果将产生重大的更广泛的科学影响，作为细胞生物学，特别是涉及干细胞的应用的使能工具。此外，抑制剂化合物可能是有吸引力的候选药物。开展的研究活动还将与教育和K-12推广计划相结合，包括在PI的实验室接待一名高中教师，并通过干细胞主题研讨会与高中生接触，以培养对干细胞研究的科学、技术、伦理和监管的普遍认识。然而，这些化合物的使用往往受到靶向性和潜在毒性不足的限制。此外，识别一种能够在细胞内环境中特异性抑制给定PPI的小分子并不是一件容易的事。为了克服这些限制，有人建议设计双环肽来特异性地抑制细胞内的PPI，其中一个环部分使细胞穿透，第二个部分选择性地与靶蛋白上的特定区域结合。在目标1中，将筛选mRNA展示文库，以确定介导细胞穿透的循环部分。在信使核糖核酸的展示中，每个多肽都与其编码序列相连。从核酸-多肽杂交物文库中选择具有所需性质的多肽，并通过DNA测序进行鉴定。在目标2中，将筛选一个包含细胞穿透部分的双环肽的mRNA展示文库，以确定能够干扰由β-连环蛋白介导的PPI的环肽部分。最后，在目标3下，双环肽抑制剂将通过在HEK293T细胞中的实验进行验证。随后，验证的抑制剂将被用于研究靶PPI在hESCs中的作用。为了促进K-12的推广，PI将接待一名高中教师作为凯南研究员计划的一部分，凯南研究员计划是北卡罗来纳州K-12公立学校教师的一个享有盛誉的一年奖学金计划，旨在促进和留住杰出的科学、技术、工程和数学(STEM)教育工作者。此外，PI将通过干细胞主题研讨会与高中生互动。拟议研究的主要方面将纳入PI目前提供的课程，本科生将积极参与有助于该项目总体目标的有针对性的研究项目。女性和少数族裔学生的参与将特别受到鼓励。该奖项由CBET部门的生物技术和生化工程项目获得，并由分子和细胞生物学部门的细胞动力学和功能项目共同资助。

1510845 Rao, Balaji M.Cellular processes such as growth, death and differentiation are regulated by interactions between proteins in the cell. Disruption of a protein-protein interaction (PPI) is a useful tool to study its significance in a given cellular context. The goal of this project is to develop a class of compounds that specifically inhibit PPIs of interest. Specifically, inhibitor compounds targeting a model PPI of relevance in cancer and human embryonic stem cells (hESCs) will be developed. If successful, the results from this project will have significant broader scientific impact as an enabling tool for cell biology, and applications involving stem cells in particular. Furthermore, inhibitor compounds may be attractive as drug candidates. Research activities undertaken will also be integrated with a plan for education and K-12 outreach, including hosting a high school teacher in the PI's lab and engaging with high school students through a stem cell themed workshop to create general awareness of the science, technology, ethics and regulation of stem cell research.Cell-permeable small molecules represent a powerful tool for inhibiting PPIs. However, the use of these compounds is often limited by insufficient target specificity and potential toxicity. Furthermore, the identification of a small molecule capable of specifically inhibiting a given PPI in the intracellular milieu is not trivial. To overcome these limitations, it is proposed to design bi-cyclic peptides for specific inhibition of intracellular PPIs, wherein one cyclic moiety enables cell penetration, and the second moiety selectively binds a specific region on the target protein. In Objective 1, mRNA display libraries will be screened to identify cyclic moieties that mediate cell penetration. In mRNA display, each peptide is linked to its coding sequence. Peptides with desired properties are selected from the library of nucleic acid-peptide hybrids and identified by DNA sequencing. In Objective 2, a mRNA-display library of bi-cyclic peptides containing a cell penetrating moiety will be screened to identify cyclic peptide moieties that disrupt a PPI mediated by beta-catenin. Finally, under Objective 3, the bi-cyclic peptide inhibitors will be validated through experiments in HEK293T cells. Subsequently, the validated inhibitor will be used to investigate the role of the target PPI in hESCs. To facilitate K-12 outreach, the PI will host a high school teacher as part of the Kenan Fellows Program - a prestigious year-long fellowship program for North Carolina K-12 public school teachers designed to advance and retain exceptional science, technology, engineering and mathematics (STEM) educators. Additionally, the PI will engage with high school students through a stem cell themed workshop. Key aspects of the proposed research will be incorporated into courses currently offered by the PI and undergraduate students will be actively involved in targeted research projects that contribute to the overall goals of this project. Participation of women and minority students will be particularly encouraged.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Cellular Dynamics and Function Program of the Division of Molecular and Cellular Biology.