MECHANISM BASED INHIBITION OF CYCLIN DEPENDENT KINASES

描述(逐字摘自申请者摘要)：细胞周期蛋白依赖性激酶 (CDK)在细胞周期调控中发挥重要作用 事件。CDK的特异性抑制剂可能被用作 阐明信号转导途径和信号转导途径的药理探针 作为细胞周期相关调控和管理的治疗剂 疾病。然而，目前大多数CDK的化学抑制剂都是作为一种 竞争性抑制剂的共同底物三磷酸腺苷往往缺乏所需 特异性和效力。这促使我们开发了特定的CDK抑制剂。 以一种新的行动机制和普遍适用的方法 蛋白质丝氨酸/苏氨酸激酶抑制剂的设计。基于机制的 使用特定底物序列设计的缓蚀剂应作为模板 提供最大概率保留模板的特异性，而不是 损害了必要的抑制效力。为了验证这一假设，有几个例子 通过掺入二肽来设计潜在的基于机理的抑制剂 甘氨酸-丝氨酸取代成已知是CDK的良好底物的序列。这个 将探索在靶向丝氨酸周围使用修饰的多肽 在将磷酸化的多肽类似物转化为更高活性的 物种，它可以与靶向激酶反应，产生共价的 灭活酶(特定目标1)。更换件的设计覆盖范围很广 为了发现所需的基本化学物质而进行的激活范围 制造CDK的“自杀”抑制剂。将对这些抑制剂进行测试 它们对重组人细胞周期蛋白B/CDC2的抑制类型和性质 激酶(特异靶向2)。将对抑制剂的特异性进行评估，并 与原始模板对细胞周期蛋白的底物特异性比较 B/CDC2与cAMP依赖的蛋白激酶和蛋白激酶C相关 (具体目标3)。未来的努力将集中在确定活跃的地点 渣油(S)的改性及提高缓蚀剂渗透性的研究 细胞膜及其对蛋白质分解的稳定性。这种方法 应普遍适用于快速发展的机构型 已知的任何蛋白丝氨酸/苏氨酸激酶的抑制剂 特定底物序列。

DESCRIPTION (verbatim from the applicant's abstract): Cyclin-dependent kinases (CDKs) play an important role in the control and regulation of cell cycle events. Inhibitors specific for CDKs could potentially be used as pharmacological probes for the elucidation of signal transduction pathways and as therapeutic agents in the control and management of cell cycle-related diseases. However, most current chemical inhibitors of CDKs act as a competitive inhibitor of the common substrate ATP and often lack the desired specificity and potency. This prompted us to develop specific CDK inhibitors with a novel mechanism of action and a generally applicable approach to the design of protein serine/threonine kinase inhibitors. Mechanism-based inhibitors designed using a specific substrate sequence, as a template should offer the best probability of retaining specificity of the template without compromising the necessary inhibitory potency. To test this hypothesis, several potential mechanism-based inhibitors are designed by incorporation of dipeptide Gly-Ser replacements into a sequence known to be a good substrate of CDKs. The use of modified peptide groups surrounding the targeted serine will be explored in converting the phosphorylated peptide analog to a more highly reactive species, which could react with the targeted kinase to produce a covalently inactivated enzyme (specific aim 1). Replacements are designed to cover a wide range of activation in order to discover the fundamental chemistry necessary for making "suicide" inhibitors of CDKs. These inhibitors will be tested for their type and nature of inhibition against a recombinant human cyclin B/CDC2 kinase (specific aim 2). The specificity of inhibitors will be evaluated and compared with the substrate specificity of the original template towards cyclin B/CDC2, relative to cAMP-dependent protein kinase and protein kinase C (specific aim 3). Future efforts will focus on identifying the active site residue(s) modified and on improving the inhibitors' permeability across cellular membranes and their stability towards proteolysis. This approach should be generally applicable to the quick development of mechanism-based inhibitors specific for any protein serine/threonine kinase with a known specific substrate sequence.