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

Richard Ågren,Tomasz Maciej Stepniewski,Kristoffer Sahlholm,Hugo Zeberg,Jana Selent

doi:10.3390/ijms22084078

Abstract

The forward (kon) and reverse (koff) 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 kons and koffs of four dopamine D2 receptor (D2R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S1935.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar koffs for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the koffs of DA and p-tyramine. Conversely, the kon 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, S1935.42A mutation lowered the kon of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic Kds derived from the koff and kon 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.

Highlights

Our results suggest that contacts with S1935.42 have a relatively greater impact on kon, compared to koff, for both DA and (S)-5-OH-DPAT, whereas the binding kinetics of p-tyramine and (R)-5-OH-DPAT were only marginally affected by S1935.42 A mutation
Following agonist application to Xenopus oocytes expressing D2 R and GIRK1/4 subunits, Gβγ subunits released from activated Gi/o protein heterotrimers open the GIRK
We used the GIRK current in oocytes voltage-clamped at −80 mV and superfused with a high-K+ buffer (25 mM KCl) as a readout of agonist binding to D2 R

Summary

Introduction

G protein-coupled receptors (GPCRs) are ubiquitously expressed in the human body and targeted by over 30% of all FDA-approved drugs [1]. Published structures of catecholamine receptors, which belong to the rhodopsin-like GPCRs ( known as class A [1]), are consistent with important polar interactions between orthosteric agonist amine groups and a conserved aspartate (D3.32 ) in the transmembrane segment (TM) 3 and between agonist electronegative groups (such as hydroxyls) and three conserved serine (S) residues in TM) 5; S5.42 , S5.43 , and S5.46 [2,3,4] (superscript numbers represent the Ballesteros–Weinstein numbering scheme [5]). D2 R is an important target for treating neurological, psychiatric, and endocrine disorders, including

Methods

Results

Conclusion