MECHANISM OF ENZYME INHIBITION BY PHARMACOLOGICAL AGENTS

本提案的目的是确定 酶和高度特异性的竞争性抑制剂。该酶 研究了哺乳动物磷酸二酯酶（PDE）的cAMP特异性同工酶， 与学习和记忆功能密切相关。抑制剂 这种酶在治疗某些 人类病理学 这项工作涉及使用一种新的体内耐药性选择 在酵母中表达的哺乳动物酶的突变体。这种重组 将哺乳动物基因产物导入酵母细胞， 系统和罕见的耐药突变体的分离机制。 所使用的选择设计用于分离哺乳动物PDE突变体 无法与竞争性抑制剂相互作用 （咯利普兰），但不影响底物（cAMP）结合或 水解 这项工作应有助于更好地了解 哺乳动物PDE酶和可用于治疗癌症药物之间的相互作用 药此外，本研究采用的方法应证明 这在设计和测试更有效的抑制剂中是有用的。 酵素该方法也将扩展到其他研究。 哺乳动物的酶这些新技术可以提供一个宝贵的工具， 用于分析各种各样的生物-酶相互作用。

The objective of this proposal is to define the interaction between an enzyme and a highly specific competitive inhibitor. The enzyme being studied is a mammalian cAMP-specific isozyme of phosphodiesterase (PDE) that is strongly implicated in learning and memory functions. Inhibitors of this enzyme are pharmacologically useful in the treatment of some human pathologies. The work involves the use of a novel in vivo drug-resistance selection for mutants of a mammalian enzyme expressed in yeast. This reconstitution of a mammalian gene product into yeast cells results in a simplified system and a mechanism for the isolation of rare drug resistant mutants. The selection to be used is designed to isolate mammalian PDE mutants that are incapable of interacting with a competitive inhibitor (rolipram), but are not affected in substrate (cAMP) binding or hydrolysis. This work should lead to a better understanding of the nature of the interaction between a mammalian PDE enzyme and a pharmacologically useful drug. In addition, the methods employed in this research should prove useful in both the design and testing of more potent inhibitors of this enzyme. The methodology will also be extended to the study of other mammalian enzymes. These new techniques could provide an invaluable tool for the analysis of a wide variety of inhibitor-enzyme interactions.