Molecular Determinants of Selectivity and Efficacy at the Dopamine D3 Receptor

Abstract

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.

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor

“…To confirm this hypothesis, D 2 -A4-mediated G protein activation was measured using a BRET assay in which agonist-bound receptor induces a separation of the G␣ energy donor (G␣ i1 -91-Rluc8) from the complemented G␤␥ acceptor (mVenus-G␤ 1 ␥ 2 ), thus decreasing the BRET signal ( Fig. 1C) (22). Activation of D 2 -WT produced a dose-dependent increase in G protein activation (Fig.…”

“…Plasmids-For the G protein activation bioluminescence resonance energy transfer (BRET) assay, pcDNA3.1 plasmids were used carrying c-Myc-tagged wild type or mutant rat D 2L (D 2 -WT, A2, A3, or A4), Renilla luciferase 8 (Rluc8) inserted at the position 91 of G␣ i1 (G␣ i1 -91-Rluc8) (21), and mVenus fragments V1 and V2 fused to G␤ 1 and G␥ 2 , respectively (V1-␤ 1 and V2-␥ 2 ) (22). For the BRET-based inhibition of cAMP assay, pcDNA3.1 plasmids carrying D2-WT or A4 and CAMYEL (ATCC) (23) were used.…”

“…For all BRET studies, constructs were transiently transfected into either HEK293 or HEK293T cells using polyethyleneimine (PEI; Polysciences, Inc., Warrington, PA), as described previously (22). The quantity of transfected DNA was adjusted so that the levels of membrane expression of wild type and mutant D 2 receptors were similar as determined by radioligand binding assay or FACS, as described below.…”

“…Emission of the donor (460 nm) and the acceptor (535 nm) was then measured once at 10 min or every 5 min for 30 min using a Victor TM X Light luminescence reader (PerkinElmer Life Sciences), and the BRET ratio was calculated as described previously (26). Cells used in the G protein activation, GRK2 recruitment to receptor, and arrestin3 recruitment to ␤2-AP-EYFP BRET assays were prepared and assayed as described previously (22) and were measured 2, 2, and 20 min, respectively, after the addition of quinpirole.…”

Is there a link between selectivity and binding thermodynamics profiles?

“…The effect of binding site waters has been recently reviewed by using WaterMap for solvation energetic calculations [11]. Selectivity between dopamine D2 and D3 receptors and kinase targets were also successfully rationalized by the analysis of binding site water molecules [12][13]. Therefore, computational approaches can significantly facilitate the design of selective compounds, if high-quality crystal structures are available.…”