A rapid screening system to determine drug affinities for the intestinal dipeptide transporter 1: system characterisation

Purpose: To establish an in vitro system for the rapid assessment of the affinities of potential substrates for the di/tri/oligopeptide transport system (DTS). Methods: Monolayers of Caco-2 cells were cultured in plastic wells for 7–9 days and the uptake of Gly-[³H]L-Pro, a specific and relatively stable substrate for the DTS was used as an affinity probe. Gly-[³H]L-Pro (50 nM), together with excess L-Pro (10 mM), to suppress uptake of any [³H]L-Pro produced by degradation of the probe, was incubated with the test compound (usually 1 mM) at pH 6 for 3 min. The uptake of radiolabel was determined by liquid scintillation counting. Results: High specific-uptake (>85%) of Gly-[³H]L-Pro was obtained with cells grown for 7–9 days. Gly-[³H]L-Pro uptake had a substantial active concentration-dependent component (K_m of 0.39±0.02 mM, V_max of 0.98±0.04 nmol min⁻¹ (mg protein)⁻¹. This process was shown to be specific for the DTS as evidenced by the significant inhibition by compounds reported to be transported by this system and the lack of inhibition by amino acids. The use of low competitor concentrations (1 mM) enabled a range of inhibition values (0–89%) of a series of competitors (amino acids, dipeptides and β-lactam antibiotics) to be estimated, illustrating that structurally similar compounds can be ranked for affinity to the DTS. Conclusion: A screening system, using Caco-2 cells and the dipeptide Gly-[³H]L-Pro as a displaceable probe, was developed to assess a variety of compounds for recognition by the di/tri/oligopeptide transport system. This fully describes the first system that allows structurally related compounds to be ranked on the basis of their affinity for the DTS recognition site.