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

ABSTRACT: 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_ons and k_offs 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 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_offs for the two 5-OH-DPAT enantiomers at wild-type (WT) D₂R, both being slower than the k_offs 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, S193^5.42A mutation lowered the k_on of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic K_ds 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.

SUBMITTER: Agren R

PROVIDER: S-EPMC8071183 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Adenosine A2A and dopamine D2 receptors in the basal ganglia form heterotetrameric structures that are involved in the regulation of motor activity and neuropsychiatric functions. The present study examines the A2A receptor-mediated modulation of D2 receptor binding in vivo using positron emission tomography (PET) with the D2 antagonist tracer [11C]raclopride. Healthy male Wistar rats (n = 8) were scanned (60 min dynamic scan) with [11C]raclopride at baseline and 7 days later following an acute administration of the A2A agonist CGS21680 (1 mg/kg), using a MicroPET Focus-220 camera. Nondisplaceable binding potential (BPND) values were calculated using a simplified reference tissue model (SRTM), with cerebellum as the reference tissue. SRTM analysis did not show any significant changes in [11C]raclopride BPND (p = 0.102) in striatum after CGS21680 administration compared to the baseline. As CGS21680 strongly affects hemodynamics, we also used arterial blood sampling and a metabolite-corrected plasma input function for compartment modeling using the reversible two-tissue compartment model (2TCM) to obtain the BPND from the k3/k4 ratio and from the striatum/cerebellum volume of distribution ratio (DVR) in a second group of animals. These rats underwent dynamic [11C]raclopride scans after pretreatment with a vehicle (n = 5), a single dose of CGS21680 (1 mg/kg, n = 5), or a single dose of the A2A antagonist KW6002 (1 mg/kg, n = 5). The parent fraction in plasma was significantly higher in the CGS21680-treated group (p = 0.0001) compared to the vehicle-treated group. GCS21680 administration significantly reduced the striatal k3/k4 ratio (p < 0.01), but k3 and k4 estimates may be less reliable. The BPND (DVR-1) decreased from 1.963 ± 0.27 in the vehicle-treated group to 1.53 ± 0.55 (p = 0.080) or 1.961 ± 0.11 (p = 0.993) after the administration of CGS21680 or KW6002, respectively. Our study suggests that the A2A agonist CGS21680, but not the antagonist KW6002, may reduce the D2 receptor availability in the striatum.

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

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

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