A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 2: affinities of ACE inhibitors

Purpose: To assess the affinities of a series of ACE inhibitors for the di/ tri/oligopeptide transport system (DTS) using a rapid in vitro system. Meth ods: Monolayers of Caco-2 cells were cultured in plastic wells for 7-9 days and the uptake of Gly-[H-3]r-Pro was used as an affinity probe. Gly-[H-3]L -Pro (50 nM), together with excess L-Pro (10 mM), to suppress uptake of any [H-3]L-Pro produced by degradation of the probe, was incubated with the te st compound (usually 1 mM) at pH 6 for 3-mins. The uptake of radiolabel was determined by liquid scintillation counting. Results: A 2-dimensional six- domain model of the transporter based on the structure of a phosphinate ACE inhibitor (SQ-29852) was constructed to facilitate interpretation of the c ompetitor affinities. The SQ-29852 molecule was divided into six binding do mains (A-F) based on functional groups within these regions and the effects of structural variation in four of these domains (A, C-E) were explored. A series of dipeptide-like compounds varying within specific domains were se lected from a large number of commercially available ACE inhibitors and SQ- 29852 analogues. Domain A had a preference for an uncharged group, with bul ky hydrophobic groups reducing affinity. Domain C exhibited a preference fo r a positive charge over a neutral function, with the space this functional group occupies contributing to affinity. Domain D favoured lipophilic resi dues and domain E retained activity when the carboxylic acid was esterified . Conclusion: The test system is able to reveal structure-activity relation ships of peptidomimetic agents and may well serve as a design tool to optim ise affinity for the DTS. (C) 2000 Elsevier Science B.V. All rights reserve d.