glycylproline and ceronapril

To establish an in vitro system for the rapid assessment of the affinities of potential substrates for the di/tri/oligopeptide transport system (DTS).. 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.. 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 (Km of 0.39 +/- 0.02 mM, Vmax 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 beta-lactam antibiotics) to be estimated, illustrating that structurally similar compounds can be ranked for affinity to the DTS.. 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.

Topics: Algorithms; Angiotensin-Converting Enzyme Inhibitors; Anti-Bacterial Agents; Caco-2 Cells; Cadherins; Carrier Proteins; Dipeptides; Drug Evaluation, Preclinical; Humans; Kinetics; Lactams; Membrane Transport Proteins; Organophosphorus Compounds; Proline; Substrate Specificity

To assess the affinities of a series of ACE inhibitors for the di/tri/oligopeptide transport system (DTS) using a rapid in vitro system.. Monolayers of Caco-2 cells were cultured in plastic wells for 7-9 days and the uptake of Gly-[3H]L-Pro 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-mins. The uptake of radiolabel was determined by liquid scintillation counting.. 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.. 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.

Topics: Algorithms; Angiotensin-Converting Enzyme Inhibitors; ATP Binding Cassette Transporter, Subfamily B, Member 2; ATP-Binding Cassette Transporters; Caco-2 Cells; Culture Media; Dipeptides; Drug Evaluation, Preclinical; Humans; Models, Biological; Organophosphorus Compounds; Proline; Structure-Activity Relationship

Oligonucleotide-based therapies represent novel strategies for manipulating the expression and function of target proteins and are undergoing clinical evaluation for the treatment of viral diseases and malignancies. However, poor biological stability and cellular delivery represent potential limitations to the therapeutic development of oligonucleotides. Conjugation of oligonucleotides to lipophilic groups can improve delivery to cells but the enhanced cellular binding may also facilitate nonspecific interactions. In this report, we show that phosphorothioate oligonucleotides conjugated to lipophilic groups, either tocopherol (Vitamin E) or 2-Di-O-hexadecyl-3-glycerol, can significantly inhibit the functioning of the dipeptide transporter system (DTS) in cultured Caco-2 intestinal cells. Because the DTS mediates the binding and absorption of nutrient peptides and important drugs, such as the cephalosporin and penicillin antibiotics, this finding has important implications in relation to the potential toxicity of lipophilic conjugates in vivo. It also suggests a potential drug interaction with lipophilic oligonucleotide-conjugates if they were to be delivered orally.

Topics: Angiotensin-Converting Enzyme Inhibitors; Biological Transport; Caco-2 Cells; Dipeptides; Dose-Response Relationship, Drug; Glyceryl Ethers; Humans; Oligodeoxyribonucleotides; Organophosphorus Compounds; Proline; Thionucleotides; Vitamin E