Dopamine D2 Receptor Agonist Binding Kinetics-Role of a Conserved Serine Residue

The forward (k(on)) and reverse (k(off)) 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 k(on)s and k(off)s of four dopamine D-2 receptor (D2R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S193(5.42) by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar k(off)s for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the k(off)s of DA and p-tyramine. Conversely, the k(on) 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, S193(5.42)A mutation lowered the k(on) of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic K(d)s derived from the k(off) and k(on) estimates correlated well with EC50 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 D2R agonist candidate drugs.

Automated summary

The study used an ion channel activation assay to estimate the rate constants of dopamine D-2 receptor agonists, with results showing higher k(on) for (S)-5-OH-DPAT compared to (R)-5-OH-DPAT, and similar k(off) for both enantiomers at wild-type D2R. Through mutagenesis, the k(on) of (S)-5-OH-DPAT can be reduced, decreasing the potency difference between the two enantiomers. The derived kinetic K(d)s correlated well with EC50 values for all compounds, indicating the assay captured meaningful information about binding kinetics.