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

Abstract 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. Methods: Monolayers of Caco-2 cells were cultured in plastic wells for 7–9 days and the uptake of Gly-[ 3 H] l -Pro was used as an affinity probe. Gly-[ 3 H] l -Pro (50 nM), together with excess l -Pro (10 mM), to suppress uptake of any [ 3 H] l -Pro produced by degradation of the probe, was incubated with the test 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 competitor affinities. The SQ-29852 molecule was divided into six binding domains (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 selected from a large number of commercially available ACE inhibitors and SQ-29852 analogues. Domain A had a preference for an uncharged group, with bulky hydrophobic groups reducing affinity. Domain C exhibited a preference for a positive charge over a neutral function, with the space this functional group occupies contributing to affinity. Domain D favoured lipophilic residues and domain E retained activity when the carboxylic acid was esterified. Conclusion: The test system is able to reveal structure-activity relationships of peptidomimetic agents and may well serve as a design tool to optimise affinity for the DTS.