Inhibitors of tripeptidyl peptidase II. 2. Generation of the first novel lead inhibitor of cholecystokinin-8-inactivating peptidase: A strategy for the design of peptidase inhibitors

The cholecystokinin-8 (CCK-8)-inactivating peptidase is a serine peptidase which has been shown to be a membrane-bound isoform of tripeptidyl peptidas e II (EC 3.4.14.10). It cleaves the neurotransmitter CCK-8 sulfate at the M et-Gly bond to give Asp-Tyr(SO3H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH2. In seek ing a reversible inhibitor of this peptidase, the enzymatic binding subsite s were characterized using a fluorimetric assay based on the hydrolysis of the artificial substrate Ala-Ala-Phe-amidomethylcoumarin. A series of di- a nd tripeptides having various alkyl or aryl side chains was studied to dete rmine the accessible volume for binding and to probe the potential for hydr ophobic interactions. From this initial study the tripeptides Ile-Pro-Ile-O H (K-i = 1 mu M) and Ala-Pro-Ala-OH (K-i = 3 mu M) and dipeptide amide Val- Nvl-NHBu (K-i = 3 mu M) emerged as leads. Comparison of these structures le d to the synthesis of Val-Pro-NHBu (K-i = 0.57 mu M) which served for later optimization in the design of butabindide, a potent reversible competitive and selective inhibitor of the CCK-8-inactivating peptidase. The strategy for this work is explicitly described since it illustrates a possible gener al approach for peptidase inhibitor design.