The dopamine D3 receptor (D3R) has been implicated in substance abuse and other neuropsychiatric disorders. The high sequence homology between the D3R and D2R, especially within the orthosteric binding site (OBS) that binds dopamine, has made the development of D3R-selective compounds challenging. Here, we deconstruct into pharmacophoric elements a series of D3R-selective substituted-4-phenylpiperazine compounds, and use computational simulations and binding and activation studies to dissect the structural bases for D3R selectivity and efficacy. We find that selectivity arises from divergent interactions within a second binding pocket (SBP) separate from the OBS, whereas efficacy depends on the binding mode in the OBS. Our findings reveal structural features of the receptor that are critical to selectivity and efficacy that can be used to design highly D3R-selective ligands with targeted efficacies. These findings are generalizable to other GPCRs in which the SBP can be targeted by bitopic or allosteric ligands.
Subtype-selective agents for the dopamine D3 receptor (D3R) have been considered as potential medications for drug addiction and other neuropsychiatric disorders. Medicinal chemistry efforts have led to the discovery of 4-phenylpiperazine derivatives that are .100-fold selective for the dopamine D3 receptor over dopamine D2 receptor (D2R), despite high sequence identity (78% in the transmembrane domain). Based on the recent crystal structure of D3R, we demonstrated that the 4-phenylpiperazine moiety in this class of D3R-selective compounds binds to the conserved orthosteric binding site, whereas the extended aryl amide moiety is oriented toward a divergent secondary binding pocket (SBP). In an effort to further characterize molecular determinants of the selectivity of these compounds, we modeled their binding modes in D3R and D2R by comparative ligand docking and molecular dynamics simulations. We found that the aryl amide moiety in the SBP differentially induces conformational changes in transmembrane segment 2 and extracellular loop 1 (EL1), which amplify the divergence of the SBP in D3R and D2R. Receptor chimera and site-directed mutagenesis studies were used to validate these binding modes and to identify a divergent glycine in EL1 as critical to D3R over D2R subtype selectivity. A better understanding of drug-dependent receptor conformations such as these is key to the rational design of compounds targeting a specific receptor among closely related homologs, and may also lead to discovery of novel chemotypes that exploit subtle differences in protein conformations.
N-(3-fluoro-4-(4-(2,3-dichloro- or 2-methoxyphenyl)piperazine-1-yl)-butyl)-aryl carboxamides were prepared and evaluated for binding and function at dopamine D3 (D3R) and D2 receptors (D2R). In this series, we discovered some of the most D3R selective compounds reported to date, (e.g. 8d and 8j >1000-fold D3R-selective over D2R.) In addition, chimeric receptor studies further identified the second extracellular (E2) loop as an important contributor to D3R binding selectivity. Further, compounds lacking the carbonyl group in the amide linker were synthesized and while these amine-linked analogues bound with similar affinities to the amides at D2R, this modification dramatically reduced binding affinities at D3R by >100-fold (e.g. D3RKi for 15b = 393 v. for 8j = 2.6 nM) resulting in compounds with significantly reduced D3R selectivity. This study supports a pivotal role for the D3R E2 loop and the carbonyl group in the 4-phenylpiperazine class of compounds and further reveals a point of separation between structure-activity relationships at D3R and D2R.
The
dopamine D3 receptor (D3R) is a promising
target for the development of pharmacotherapeutics to treat substance
use disorders. Several D3R-selective antagonists are effective
in animal models of drug abuse, especially in models of relapse. Nevertheless,
poor bioavailability, metabolic instability, and/or predicted toxicity
have impeded success in translating these drug candidates to clinical
use. Herein, we report a series of D3R-selective 4-phenylpiperazines
with improved metabolic stability. A subset of these compounds was
evaluated for D3R functional efficacy and off-target binding
at selected 5-HT receptor subtypes, where significant overlap in SAR
with D3R has been observed. Several high affinity D3R antagonists, including compounds 16 (Ki = 0.12 nM) and 32 (Ki = 0.35 nM), showed improved metabolic stability
compared to the parent compound, PG648 (6). Notably, 16 and the classic D3R antagonist SB277011A (2) were effective in reducing self-administration of heroin
in wild-type but not D3R knockout mice.
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