Abstract
A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D2 receptor (D2R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D2R antagonism by a series of aripiprazole analogues, as well as the recovery of dopamine (DA) responsivity upon washout. The aripiprazole analogues comprise an orthosteric and a secondary pharmacophore and differ by the length of the saturated carbon linker joining these two pharmacophores. Two compounds containing 3- and 5-carbon linkers allowed for a similar extent of recovery from antagonism in the presence of 1 or 100 μM DA (>25 and >90% of control, respectively), whereas recovery was less prominent (∼20%) upon washout of the 4-carbon linker compound, SV-III-130, both with 1 and 100 μM DA. Prolonging the coincubation time with SV-III-130 further diminished recovery. Curve-shift experiments were consistent with competition between SV-III-130 and DA. Two mutations in the secondary binding pocket (V91A and E95A) of D2R decreased antagonistic potency and increased recovery from SV-III-130 antagonism, whereas a third mutation (L94A) only increased recovery. Our results suggest that the secondary binding pocket influences recovery from inhibition by the studied aripiprazole analogues. We propose a mechanism, supported by in silico modeling, whereby SV-III-130 initially binds reversibly to the D2R, after which the drug-receptor complex undergoes a slow transition to a second ligand-bound state, which is dependent on secondary binding pocket integrity and irreversible during the time frame of our experiments.
Highlights
The action of angiotensin-1 receptor antagonists is prolonged by their long-lasting, induced-fit binding to their target receptor, reflected by insurmountable antagonism in in vitro experiments.[8]
To determine the influence of linker length on the kinetics, reversibility, and surmountability of D2 receptor (D2R) antagonism, we studied the properties of the three aripiprazole analogues, SWR-1-8, SV-III-130, and SWR-1-14, in antagonizing DA
We found that L94A mutation creates an empty space adjacent to transmembrane segment 2 which becomes occupied by W100, enabling tight packing of W100 against transmembrane segment 2 (Figure 5D)
Summary
The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) and an important pharmaceutical target.D2R antagonism or weak partial agonism is the common denominator of antipsychotic mainstay of Parkinson’s disease dtrreuagtsm, eanntd.1−D42TRheagtoimniesmcouisrsea of receptor occupancy has been suggested to have an important impact on the clinical properties of therapeutic ligands, including antipsychotics, where transient rather than continuous D2R occupancy may be associated with a more favorable profile in terms of extrapyramidal side effects.[5]Ligand binding kinetics is presumed to play an important role in determining the time course of occupancy at the target receptor.[6,7] For example, the action of angiotensin-1 receptor antagonists is prolonged by their long-lasting, induced-fit binding to their target receptor, reflected by insurmountable antagonism in in vitro experiments.[8]. The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) and an important pharmaceutical target. D2R antagonism or weak partial agonism is the common denominator of antipsychotic mainstay of Parkinson’s disease dtrreuagtsm, eanntd.1−D42TRheagtoimniesmcouisrsea of receptor occupancy has been suggested to have an important impact on the clinical properties of therapeutic ligands, including antipsychotics, where transient rather than continuous D2R occupancy may be associated with a more favorable profile in terms of extrapyramidal side effects.[5]. Ligand binding kinetics is presumed to play an important role in determining the time course of occupancy at the target receptor.[6,7] For example, the action of angiotensin-1 receptor antagonists is prolonged by their long-lasting, induced-fit binding to their target receptor, reflected by insurmountable antagonism in in vitro experiments.[8] Insurmountable antagonism is characterized by a decrease in the maximal agonistinduced response, which cannot be overcome by increasing the concentration of agonist.[8]. Receptor binding kinetics has been predicted to be an important determinant of positron emission tomography (PET) tracer characteristics, such as sensitivity to endogenous neurotransmitter release.[12]
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