MECHANISM OF ENZYME INHIBITION BY PHARMACOLOGICAL AGENTS

本提案的目标是定义 酶和一种高度特异的竞争性抑制物。这种酶是 研究的是哺乳动物磷酸二酯酶(Pde)的cAMP特异性同工酶。 这与学习和记忆功能密切相关。抑制剂 这种酶对治疗某些疾病有药理上的作用 人类的病理学。 这项工作涉及到使用一种新型的体内抗药性选择 在酵母中表达的一种哺乳动物酶的突变体。这次重组 将哺乳动物基因产物导入酵母细胞，结果是简化了 用于分离罕见耐药突变株的系统和机制。 要使用的选择是为了分离哺乳动物的PDE突变体 它们不能与竞争性抑制物相互作用 (罗利普兰)，但不影响底物(CAMP)结合或 水解液。 这项工作应该有助于更好地理解 哺乳动物PDE酶与一种药理有用物质之间的相互作用 毒品。此外，这项研究中使用的方法应该证明 在设计和测试更有效的这种抑制药方面都很有用 酵素。该方法也将扩展到对其他 哺乳动物的酶。这些新技术可以提供一个无价的工具 用于分析各种抑制剂-酶的相互作用。

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.