The Identification and Characterization of Synthetic Peptide Substrates and Inhibitors for Protein Kinases

合成肽底物和蛋白激酶抑制剂的鉴定和表征

• 批准号: 9728399
• 负责人: Kit Lam
• 金额: $ 9.2万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 1999-07-08
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9728399&HistoricalAwards=false
• 关键词: Identification; Characterization; Synthetic; Peptide; Substrates

Lam 9728399 1. Technical This study focuses on protein tyrosine kinases (PTKs), which play an important role in signal transduction for a broad spectrum of cellular activities, including T-cell and B-cell activation, responses to extracellular stimuli, mitogenesis, differentiation, and oncogenesis. The objective is to further our understanding of the range and substrate specificity of these enzymes, particularly peptide substrates and inhibitors for p60c-src PTK. The structure-activity relationship, kinetic parameters, physico-chemical interaction, and biological and biochemical effects on v-src transfected cells or cells that have constitutively elevated c-src activity are basic issues that are studied. Experiments include: evaluation "one-bead one-compound" combinatorial peptide libraries with p60c-src PTK for identification of additional substrate motifs; design of potent pseudosubstrate-based peptide inhibitors for p60c-src PTK; detailed kinetic studies on the identified substrates and inhibitors; probing the biochemical effects of the peptide inhibitors on the phosphorylation of cellular proteins; examining the biological and biochemical effects of the peptide inhibitors on v-src transfected 3T3 and other cell lines; computer modelling ( DOCK program) the interaction between the peptide substrates or inhibitors with the known crystal structure of p60c-src PTK; and determining the X-ray crystal structure of the co-crystal (in collaboration with Professor Stephen Harrison and Michael Eck of Harvard University). 2. Non-technical Protein tyrosine kinases represent a class of enzymes that play extremely important roles in various cellular activities such as cell division, cell death, and cancer formation. Despite their great importance, very little is known about the mechanism of action of these enzymes. The objective of this study is to build on what has already been discovered in this laboratory in the last two years (NSF Grant MCB 9506217) on the p60c-src protein tyrosine ki nase project. The peptide substrates and inhibitors that have already been identified will be used to further study the mechanism of action of this enzyme. Kinetic studies will also be performed with these peptides. Additionally, Dr. Lam has established a collaboration with Drs. Eck and Harrison of Harvard University to determine the X-ray structure of the peptide-enzyme complex. Also planned is the use of computer modelling techniques to understand how these peptides interact with the enzyme. Finally, different approaches will be followed to deliver the peptide inhibitors into cells and study their fundamental biological effect on cell lines which have an elevated level of protein for p60c-src tyrosine kinase.

Lam 9728399 1.技术 本研究的重点是蛋白酪氨酸激酶（PTKs），它在信号转导中发挥重要作用，广泛的细胞活动，包括T细胞和B细胞活化，对细胞外刺激的反应，有丝分裂，分化和肿瘤发生。目的是进一步了解这些酶的范围和底物特异性，特别是p60 c-src PTK的肽底物和抑制剂。 构效关系、动力学参数、理化相互作用以及对v-src转染细胞或c-src活性组成性升高的细胞的生物学和生化效应是研究的基本问题。 实验包括：用p60 c-src PTK评估“一珠一化合物”组合肽文库以鉴定额外的底物基序;设计p60 c-src PTK的有效的基于假底物的肽抑制剂;对鉴定的底物和抑制剂进行详细的动力学研究;探测肽抑制剂对细胞蛋白磷酸化的生物化学作用;检测肽抑制剂对v-src转染的3 T3和其它细胞系的生物学和生物化学作用;计算机建模（DOCK程序）肽底物或抑制剂与p60 c-src PTK的已知晶体结构之间的相互作用;并确定共晶的X射线晶体结构（与哈佛大学的Stephen Harrison教授和Michael Eck合作）。 2.非技术 蛋白酪氨酸激酶代表一类在各种细胞活动如细胞分裂、细胞死亡和癌症形成中发挥极其重要作用的酶。 尽管它们非常重要，但对这些酶的作用机制知之甚少。 本研究的目的是建立在过去两年（NSF Grant MCB 9506217）在该实验室中已经发现的p60 c-src蛋白酪氨酸激酶项目的基础上。 已经鉴定的肽底物和抑制剂将用于进一步研究这种酶的作用机制。 还将对这些肽进行动力学研究。 此外，Lam博士还与哈佛大学的Eck和Harrison博士建立了合作关系，以确定肽-酶复合物的X射线结构。 还计划使用计算机建模技术来了解这些肽如何与酶相互作用。 最后，将遵循不同的方法将肽抑制剂递送到细胞中，并研究它们对具有升高的p60 c-src酪氨酸激酶蛋白水平的细胞系的基本生物学效应。