Abstract

Introduction. The binding kinetics of ligands,
described by their association (k_on) and dissociation (k_off)
rate constants at the target receptor, significantly influence drug action. For
example, recent studies on the D2 dopamine receptor demonstrate links between agonist
kinetics and observed signalling bias [1], and correlate antipsychotic side
effects with association rates and their potential influence on rebinding [2].
The D1 receptor (D1R) offers an alternate target in ameliorating symptoms of schizophrenia
[3], and while functional effects of D1R antagonists have previously been
assessed [3], less is known about their binding kinetics. Here we develop a
time-resolved fluorescence (HTRF) D1R binding assay, and demonstrate its
applicability in measuring antagonist kinetic parameters by the competition
association method [4].

Methods. Membranes expressing
Terbium-labelled SNAP-tagged human dopamine D1 receptor were generated as
previously described [2]. Binding assays were carried out using SKF83566-green
(F-SKF) as the fluorescent tracer acceptor (with HTRF measured on a BMG
Pherastar FSX). F-SKF k_on and k_off were determined by
global fitting of a one-site model (Graphpad Prism) to its association kinetics
at different tracer concentrations.  Competition
association kinetic assays were initiated by adding membranes to a mix of F-SKF
(300nM) and increasing concentrations of unlabelled antagonist, from which
competing ligand k_on and k_off were determined by the Motulsky-Mahan
(MM) model [4].

Results. The F-SKF k_on
was 3.17 ± 0.12x10⁶ M^-1min^-1, its k_off
0.41 ± 0.01min^-1, and equilibrium dissociation constant K_D 139.1 ± 5.0nM
(n=4), measured at the D1R. Using the MM approach, the kinetics of
representative D1 antagonists SCH-23390, ecopipam (SCH-39166), and clozapine
were also derived (n=3). SCH-23390 and ecopipam had similar k_on estimates
(8.89 ± 1.01 x 10⁷ and 8.82 ± 0.63 x 10⁷ M^-1
min^-1 respectively), while clozapine had a slower on-rate (1.68 ± 0.25
x 10⁷). While SCH-23390 and ecopipam again showed similar k_off
values (0.21 ± 0.004 and 0.35 ± 0.02 min^-1 respectively), off-rates for clozapine
was markedly faster (5.96 ± 0.61 min^-1).

Conclusion. HTRF screening using the F-SKF
tracer offers a robust method to assess antagonist binding kinetics at D1R,
with a homogenous, medium throughput format allowing implementation of such
studies at an early stage in compound profiling.

References

[2]
Sykes DA et. al., (2017) Extrapyramidal side effects of antipsychotics
are linked to their association kinetics at dopamine D2 receptors. Nat Commun.
2;8(1):763.

[3]
Beaulieu JM, Gainetdinov RR (2011) The physiology, signaling and pharmacology
of dopamine receptors. Pharmacol Rev, 63(1):182-217.

[4]
Motulsky HJ, Mahan LC (1984) The kinetics of competitive radioligand binding
predicted by the law of mass action. Mol Pharmacol. 25(1):1-9.