Dopamine D Receptor Agonist Binding Kinetics-Role of a Conserved Serine Residue.

The forward (k) and reverse (k) rate constants of drug-target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the ks and ks of four dopamine D receptor (DR) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S193 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar ks for the two 5-OH-DPAT enantiomers at wild-type (WT) DR, both being slower than the ks of DA and p-tyramine. Conversely, the k of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S193A mutation lowered the k of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic Ks derived from the k and k estimates correlated well with EC values for all four compounds across four orders of magnitude, strengthening the notion that our assay captured meaningful information about binding kinetics. The approach presented here may thus prove valuable for characterizing DR agonist candidate drugs.