Identification of a Potent Human Trace Amine-Associated Receptor 1 Antagonist

Abstract

Human trace amine-associated receptor subtype 1 (hTAAR1) is a G protein-coupled receptor that has therapeutic potential for multiple diseases, including schizophrenia, drug addiction, and Parkinson's disease (PD). Although several potent agonists have been identified and have shown positive results in various clinical trials for schizophrenia, the discovery of potent hTAAR1 antagonists remains elusive. Herein, we report the results of structure-activity relationship studies that have led to the discovery of a potent hTAAR1 antagonist (RTI-7470-44, 34). RTI-7470-44 exhibited an IC₅₀ of 8.4 nM in an in vitro cAMP functional assay, a K_i of 0.3 nM in a radioligand binding assay, and showed species selectivity for hTAAR1 over the rat and mouse orthologues. RTI-7470-44 displayed good blood-brain barrier permeability, moderate metabolic stability, and a favorable preliminary off-target profile. Finally, RTI-7470-44 increased the spontaneous firing rate of mouse VTA dopaminergic neurons and blocked the effects of the known TAAR1 agonist RO5166017. Collectively, this work provides a promising hTAAR1 antagonist probe that can be used to study TAAR1 pharmacology and the potential therapeutic role in hypodopaminergic diseases such as PD.

Keywords: Parkinson’s disease; cAMP functional assay; dopaminergic neurons; spontaneous firing rate; structure−activity relationship; trace amine-associated receptor 1 antagonist.

Figure 1.

TAAR1 antagonists in the literature.

Figure 2.

Compound 1 structure and key areas for medicinal chemistry SAR.

Scheme 1.

Synthesis of lead compound 34. Reagents and conditions: (a) K₂CO₃ (2 equiv), DCM, rt, 4 h; (b) cyanothioacetamide (1.5 equiv), DABCO (1 equiv), EtOH, reflux, 3 h; (c) KOH (1 equiv), DMF, rt, 12 h.

Figure 3.

Activity profile of 34. (A) Antagonist activity of 34 in hTAAR1 cAMP IC₅₀ assay. (B-C) Schild analysis to determine mode of antagonism. Each data point is the mean ± S.E.M. of three independent experiments conducted in duplicate.

Figure 4.

EPPTB or compound 34 increases firing of VTA dopamine neurons in mice. (A) Sample recordings of spontaneous firing before and after a 5 min application of compound 34 (40 μM) in dopamine neurons. (B) Time course of application of 40 μM compound 34 (N/n=7/3). (C) Compound 34 increased firing of dopamine neurons from baseline at 10–40 μM (one sampled t-tests 0.1 μM: t(6) = 1.7, P = 0.2, N/n=7/3; 10 μM: t(7) = 4.5, P = 0.003**, N/n=8/4; 40 μM: t(13) = 2.8, P = 0.01*, N/n=14/6). (D) Sample recordings of spontaneous firing before and after a 10 min application of RO5166017 (0.5 μM) and then RO5166017 with compound 34 (40 μM). (E) Time course of application of RO5166017 (0.5 μM) and then RO5166017 with compound 34 (40 μM). (F) RO5166017 (0.5 μM) suppressed firing rate of dopamine neurons and application of compound 34 reversed the inhibitory effects of RO5166017 (RM one way ANOVA, F(1.8, 10.9) = 12.65, P =0.0017; Dunnett’s multiple comparison test: Baseline vs RO5166017, P = 0.0096**, baseline vs compound 34 + RO5166017, P = 0.99, N/n=7/3). (G) Sample recordings of spontaneous firing before and after a 5 min application of 10 nM EPPTB and EPPTB with compound 34 (40 μM) in dopamine neurons. (H) Time course of application of 10 nM EPPTB and then EPPTB with compound 34 (40 μM). (I) EPPTB increased firing of dopamine neurons from baseline, but co-application of EPPTB and compound 34 decreased firing (RM One way ANOVA, F(1.3, 7.7) = 16.20, P = 0.003; Dunnett’s multiple comparison test: Baseline vs EPPTB, P = 0.0012**, baseline vs. EPPTB + compound 34, N/n=7/3, P = 0.38). (J) Sample recordings of spontaneous firing before and after a 5 min application of compound 34 (40 μM) and then compound 34 with 10 nM EPPTB in dopamine neurons. (K) Time course of application of compound 34 (40 μM) and then compound 34 with 10 nM EPPTB. (L) Co-application of compound 34 and EPPTB increased firing from baseline (RM one way ANOVA, F(1.4, 8.6) = 10.3, P = 0.0077; Dunnett’s multiple comparison test: Baseline vs compound 34, P = 0.0069**, baseline vs compound 34 + EPPTB, P = 0.016*). Vertical shaded bars represent averaged periods analyzed for the bar graph. Bars are mean ± S.E.M and symbols represent responses from individual neurons.