CAREER:Yeast-based disulfide trapping for engineering selective inhibitors of a protein kinase

这项由生物技术、生物化学和生物质工程项目颁发的国家科学基金奖支持Park教授实验室设计和表征新型Erk-2激酶肽抑制剂的工作。Erk-2在细胞发育、增殖和恶性肿瘤中发挥作用，是细胞中磷酸化依赖性信号传导的重要模型系统。考虑到哺乳动物基因组编码大量参与磷酸化调节的酶，能够特异性靶向选定激酶的活性应该会导致更好的细胞内通讯模型以及更有效的治疗由过度活跃的激酶引起的遗传疾病。该提案旨在通过结合酵母展示和二硫捕获来设计高亲和力，高特异性的Erk-2肽/蛋白抑制剂。设计的抑制剂将为小分子药物之外的激酶调节提供正交解决方案，并显著增加调节激酶途径的候选分子的潜在库。设计的肽抑制剂有望比小分子抑制剂具有更有利的生物物理特性，包括i)更高的相互作用特异性，以减少不必要的副作用，以及ii)对靶酶偶然突变的稳健性，以尽量减少耐药的发生。所提出的二硫化物捕获策略，已被证明是交联相互作用的蛋白质在同一个酵母细胞中共表达，提供了一个独特的优势，比传统的筛选方法，相互作用的特异性是内在构建在筛选，因此鉴定克隆更有可能是功能相关的。因此，该协议的成功实施将与其他特定蛋白质相互作用的工程研究高度相关。朴教授一直积极参与布法罗大学各种旨在鼓励和留住少数民族学生的外展项目。这笔资金将支持他继续参与大学、学院和院系层面的教育活动，并为发起其他包容性项目创造场所，使社区和大学更紧密地联系在一起。此外，将根据所述研究开发新的教学材料，用于本科化学和生物工程课程。该建议的教育部分将在多个层面上共同提高教育质量，同时为公众提供科学的管理。

This NSF award by the Biotechnology, Biochemical and Biomass Engineering program supports work in Prof. Park's lab to engineer and characterize novel peptide inhibitors of the Erk-2 kinase. Erk-2 plays a role in cell development, proliferation, and malignancies, and is an important model system for phosphorylation-dependent signaling in the cell. Considering that the mammalian genome encodes a large number of enzymes involved in phosphorylation regulation, being able to specifically target the activity of select kinases should lead to a better model of intracellular communication as well as to more effective therapeutics against genetic diseases caused by overactive kinases. The proposal seeks to engineer high affinity, high specificity peptide/protein inhibitors of Erk-2 by combining yeast display with disulfide trapping. The designed inhibitors will provide an orthogonal solution to kinase regulation beyond small molecule drugs, and significantly increase the potential pool of candidate molecules for regulating the kinase pathways. The designed peptide inhibitors are expected to have more favorable biophysical properties than small molecule inhibitors, including i) higher specificity of interaction to reduce unwanted side effects, and ii) robustness against fortuitous mutations in the target enzyme to minimize the occurrence of drug resistance. The proposed disulfide trapping strategy, which has been shown to crosslink interacting proteins that are co-expressed in the same yeast cell, provides a unique advantage over conventional screening methods in that the specificity of interaction is intrinsically built in the screen, so that the identified clones are far more likely to be functionally relevant. The successful implementation of the protocol will therefore be highly relevant to other engineering studies of specific protein interactions. Prof. Park has been an active participant of various outreach programs at UB designed to encourage and retain minority students in science and engineering. The funding will support his continued engagement in educational activities initiated at the university, school and department levels, as well as create venues to initiate other inclusive programs to bring the community and university closer together. Additionally, new pedagogical materials will be developed based on the described research for use in undergraduate chemical and biological engineering classes. The educational components of the proposal will collectively improve the quality of education at multiple levels, while providing scientific stewardship to the public.