Ligand with Two Modes of Interaction with the Dopamine D₂ Receptor–An Induced-Fit Mechanism of Insurmountable Antagonism

Abstract

A solid understanding of the mechanisms governing ligand binding is crucial for rational design of therapeutics targeting the dopamine D 2 receptor (D 2 R). Here, we use G protein-coupled inward rectifier potassium (GIRK) channel activation in Xenopus oocytes to measure the kinetics of D 2 R 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 D 2 R 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 D 2 R, 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.

Evidence for Two Modes of Binding of the Negative Allosteric Modulator SB269,652 to the Dopamine D2 Receptor

“…Those previous studies used pre-incubation protocols, where SB269,652 was applied prior to DA. Our recent investigation of a distinct bivalent D 2 R ligand revealed evidence of an induced-fit binding mode, where initially rapidly reversible binding becomes virtually irreversible over the time course of a few minutes [ 9 ]. We therefore investigated whether the order of the application of SB269,652 and DA could have an impact on the inhibitory potency of the former ligand.…”

“…Previous investigations have demonstrated the D 2 R secondary binding pocket to confer induced-fit binding of bitopic ligands, with mutation of SBP residues increasing ligand dissociation rates [ 9 , 18 ]. In addition, the SBP has been shown to play an important role in the allosteric pharmacology of SB269,652 [ 7 , 11 ].…”

“…At the D 2 R L94A mutant, pre-application of SB269,652 provided a 6.8-fold more potent inhibition compared to the co-application condition ( Figure 2 D; Table 2 ), whereas mutating the residue E95 to alanine reduced the potency shift observed upon pre-application to 2.2-fold ( Figure 2 E; Table 2 ). In experiments with the W100A mutant D 2 R, 100 nM DA was used to elicit GIRK activation, to compensate for the previously observed [ 9 ] ~10-fold higher EC 50 of DA at this mutant receptor compared to WT ( Figure 2 B; Table 1 ). Pre-application of SB269,652 resulted in a 102.3-fold leftward shift in pIC 50 at this mutant ( Figure 2 F; Table 2 ).…”

“…The affinities, efficacies, and (when applicable) allosteric properties of several D 2 / 3 R ligands encompassing both a primary and a secondary pharmacophore, including SB269,652, have been demonstrated to be dependent on the length of the linker joining these two pharmacophores [ 9 , 10 ]. Furthermore, mutations in TM2 of the D 2 R were shown to reduce negative cooperativity of SB269,652, indicating a role for the SBP in the allosteric actions of this ligand [ 7 ].…”

“…Taking advantage of the temporal resolution afforded by a GIRK channel activation assay, we recently demonstrated that the aripiprazole analogue SV-III-130 interacts with D 2 R by way of an induced-fit mechanism leading to the irreversible binding of this bivalent ligand. Furthermore, this behavior was observed to be crucially dependent on TM2 and ECL1 [ 9 ]. Here, using the same assay system, we investigated the influence of the OBS and SBP on the ability of SB269,652 to negatively modulate D 2 R activation by DA.…”