Synthesis and pharmacological validation of a novel radioligand for the orphan GPR88 receptor

Abstract

GPR88 is an orphan G protein-coupled receptor which has been implicated in a number of striatal-associated disorders. Herein we describe the synthesis and pharmacological characterization of the first GPR88 radioligand, [³H]RTI-33, derived from a synthetic agonist RTI-13951-33. [³H]RTI-33 has a specific activity of 83.4 Ci/mmol and showed one-site, saturable binding (K_D of 85 nM) in membranes prepared from stable PPLS-HA-hGPR88-CHO cells. A competition binding assay was developed to determine binding affinities of several known GPR88 agonists. This radioligand represents a powerful tool for future mechanistic and cell-based ligand-receptor interaction studies of GPR88.

Graphical Abstract

GPR88 is a Gα_i/o-coupled orphan G protein-coupled receptor (GPCR) that has robust expression in both striatonigral and striatopallidal pathways of the striatum.^1-3 Studies in both animals and humans support the premise that GPR88 is a novel drug target for the treatment of several central nervous system (CNS) disorders such as Parkinson’s disease, schizophrenia, drug addiction, and attention-deficit/hyperactivity disorder (ADHD). A number of studies using GPR88 knockout (KO) mice demonstrated that genetic ablation of GPR88 induced a state of hypersensitivity to the dopamine system and produced multiple behavioral phenotypes including disrupted prepulse inhibition of startle response, motor hyperactivity, poor motor coordination and balance, improved spatial learning, and low anxiety.^4-10 Recent studies also demonstrated that GPR88 KO mice had increased alcohol drinking and seeking behaviors and showed higher motor impulsivity and reduced attention, which link GPR88 to the risk of alcoholism and ADHD.^{11, 12} Moreover, human genetic studies have found positive association between the Gpr88 gene and several psychiatric disorders including schizophrenia, bipolar disorder, childhood speech delay, learning disabilities, and chorea.^{13, 14} Given the potential therapeutic applications of GPR88, elucidating the signaling mechanisms, pharmacology and function of the receptor are of importance for both basic orphan GPCR research and drug discovery. In this regard, radioligand binding is a gold standard method but is currently unavailable for GPR88 research.

To date, the endogenous ligand for GPR88 has not been discovered. In order to characterize GPR88 signaling mechanisms and biological functions, our laboratory, as well as others, has carried out a medicinal chemistry campaign to develop GPR88 synthetic agonists.^15-24 These agonists are mainly based on two scaffolds: 2-PCCA [(1R,2R)-2-(pyridin-2-yl)cyclopropane carboxylic acid ((2S,3S)-2-amino-3-methylpentyl)-(4’-propylbiphenyl-4-yl)amide (1)] and 2-AMPP [(2S)-N-((1R)-2-amino-1-(4-(2-methylpentyloxy)-phenyl)ethyl)-2-phenylpropanamide (3), Figure 1]. We recently developed a potent, selective, and brain-penetrant GPR88 agonist, RTI-13951-33 (2), that we deemed suitable for radiolabeling to study ligand-receptor interactions due to its high specific binding for the target receptor and low non-specific binding to other endogenous binding sites. RTI-13951-33 had an EC₅₀ of 45 nM in an in vitro cAMP functional assay and significantly increased [³⁵S]GTPγS binding (EC₅₀ = 535 nM) in mouse striatal membranes but not in membranes from GPR88 KO mice even at a concentration of 100 μM, indicating the compound has GPR88-specific agonist signaling activity in the striatum.²¹ In addition, the compound had no significant binding affinities at over 60 CNS targets, including GPCRs, ion channels, and neurotransmitter transporters when screened at Eurofins Panlabs²¹ and the NIMH Psychoactive Drug Screening Program (Supporting Information Table 1). RTI-13951-33 is highly water-soluble, has a low lipophilicity (clogP 3.34) suggesting the potential for low non-specific binding, and can penetrate into the brain in the pharmacokinetic studies.²¹ Notably, RTI-13951-33 significantly reduced alcohol intake and preference in wild-type mice but not in GPR88 KO mice, suggesting a specific GPR88-mediated effect on alcohol drinking.²⁵ Collectively, the favorable physiochemical properties and pharmacology profile make RTI-13951-33 a suitable ligand for radiolabeling and receptor binding studies of GPR88. Herein, we describe the synthesis and pharmacological validation of the first radioligand for GPR88, [³H]RTI-33, derived from RTI-13951-33.

Figure 1.

Selected GPR88 agonists.

[³H]RTI-33 (7) was prepared following the procedure depicted in Scheme 1. Alcohol 5^{18, 21} was treated with sodium hexamethyldisilazide (NaHMDS) in N,N-dimethylformamide (DMF) solution to generate the alkoxide anion. Commercially-available tritium-labelled methyl nosylate ([³HMeONs]) was concentrated from the supplied ethyl acetate and hexane solution, then reconstituted as a solution in DMF and transferred into the 5 alkoxide solution. The room temperature reaction was monitored by HPLC with radioactivity flow-monitoring until the reaction was complete. The intermediate 6 was purified by preparative HPLC, then stored as a solution in ethanol until needed. To afford the desired 7, a sample of the ethanol solution of 6 was evaporated under a stream of nitrogen then treated with hydrogen chloride in methanol. The methanol solution was similarly evaporated. The sample was reconstituted in ethanol solution to give [³H]RTI-33 (7) with a radiochemical purity of 98% and specific activity of 83.4 Ci/mmol.

Scheme 1.

Reagents and conditions: (a) NaHMDS, [³H]methyl nosylate, DMF, rt; (b) HCl, CH₃OH, rt.

We characterized the synthesized [³H]RTI-33 to establish a pharmacological binding profile²⁶ by carrying out saturation and competition binding studies using membranes prepared from our stable PPLS-HA-hGPR88-CHO cells, which have been used to characterize GPR88 ligands in cAMP assays.^{18, 20-23} Saturation binding studies were used to determine the affinity (K_D) of the radioligand, as well as the binding site density (B_max) using filtration-based methods. In this assay, 8 different radioligand concentrations ranging from 0.1 to 450 nM were tested in the presence of vehicle (total binding) or 10 μM RTI-13951-33 (non-specific binding, NSB). One-site specific [³H]RTI-33 binding was observed with K_D = 85 nM and B_max = 4.5 pmol/mg protein (Figure 2). The in vitro cAMP EC₅₀ value of unlabeled RTI-13951-33 is 45 nM, which compares well with the K_D of 85 nM.

Figure 2.

Saturation curve for one-site specific [³H]RTI-33 binding to PPLS-HA-hGPR88-CHO membranes using filtration methods. Data points are the means ± SEM of three independent experiments, each performed with duplicate determinations. Inset: Scatchard plot of the same data.

Next, we performed competition binding studies using analogs from both the 2-PCCA^{15, 18, 21} and 2-AMPP²⁰ scaffold families to characterize the ligand recognition properties of the radioligand binding site and to determine whether the binding affinity (K_i) correlates with the functional activity (Figure 3). Three analogs are from the 2-PCCA family [2-PCCA (1), (S,S)-2-PCCA, and RTI-13951-33 (2)] and two analogs are from the 2-AMPP family [2-AMPP (3) and 4, Figure 1]. These assays were run with 70 nM of [³H]RTI-33 because the NSB at this concentration amounted to 41% of the total binding and was thus in a tolerable range.^{27, 28} Compound K_i values are presented in Table 1 along with the in vitro functional cAMP EC₅₀ values. The K_i/EC₅₀ ratio was calculated to track the difference between binding affinity and functional activity.

Figure 3.

Competition binding of selected GPR88 agonists with [³H]RTI-33 in PPLS-HA-hGPR88-CHO membranes using filtration methods. Data points are the means ± SEM of three independent experiments, each performed with duplicate determinations. (A) Competition binding of RTI-13951-33, 2-PCCA, and (S,S)-2-PCCA. (B) Competition binding of 2-AMPP and compound 4.

Table 1.

Comparison of binding affinities and in vitro functional EC₅₀ values for GPR88 agonists.

Compound	cAMP EC50 (nM)	Ki (nM)a	Ki/EC50 Ratio
RTI-13951-33 (2)	45	224 ± 36	5.0
2-PCCA (1)	74	277 ± 39	3.7
(S,S)-2-PCCA	1738	487 ± 46	0.3
2-AMPP (3)	304	219 ± 54	0.7
4	195	612 ± 86	3.1

^aK_i values are the means ± SEM of at least three independent experiments, each performed with duplicate determinations.

Unlabeled RTI-13951-33 has a binding affinity of 224 nM, a functional potency of 45 nM, and a K_i/EC₅₀ ratio of 5.0. Compared to RTI-13951-33, 2-PCCA has a 1.2-fold lower affinity (K_i = 277 nM), a 1.6-fold lower functional potency (EC₅₀ = 74 nM), and a slightly smaller K_i/EC₅₀ ratio of 3.7. Surprisingly, although the (S,S)-isomer of 2-PCCA (EC₅₀ = 1738 nM) is 23-fold less potent than 2-PCCA (EC₅₀ = 74 nM), it has a moderate binding affinity (K_i of 487 nM) at GPR88. This suggests that the compound has a stronger affinity for the GPR88 receptor compared to its ability to functionally activate the receptor. Note: the (R,R)-isomer and (S,S)-isomer are differentiated only at two chiral centers of the cyclopropane ring, whereas the side chains in both isomers are derived from L-isoleucine (Figure 1). In general, the rank order of the binding affinities of 2-PCCA analogs is consistent with that of EC₅₀ values in the functional cAMP assay. Two compounds from the 2-AMPP series were also examined. 2-AMPP has a K_i = 219 nM, which is similar to its cAMP potency (EC₅₀ = 304 nM). On the other hand, compound 4 has a K_i = 612 nM, which is 3.1-fold lower that its functional potency. Overall, these results indicate that all functional GPR88 agonists tested in this study were able to displace [³H]RTI-33 in a competitive manner. These results also validate our radioligand for use in determining binding affinities of potential GPR88 ligands. Recently, a cryogenic electron microscopy (cryo-EM) structure of GPR88 in complex with 2-PCCA was reported.²⁹ Interestingly, 2-PCCA is an allosteric agonist binding to a pocket formed by transmembrane segments 5, 6 and the C terminus of the α5 helix of the G_i1 protein. [³H]RTI-33 radioligand binding, in combination with site-directed mutagenesis studies, will further our understanding of the GPR88 ligand binding sites and possibly mechanisms of activation of this novel receptor.

Finally, we determined the affinity (K_D) of the radioligand, as well as the binding site density (B_max) in native tissue by performing saturation binding in striatal membranes prepared from wild-type mice.²¹ In this assay, 8 different radioligand concentrations ranging from 0.1 to 250 nM were tested in the presence of vehicle (total binding) or 10 μM RTI-13951-33 (NSB). One-site specific [³H]RTI-33 binding was observed with K_D = 41 nM and B_max = 15 pmol/mg protein (Figure 4). The K_D of [³H]RTI-33 in mouse striatal membranes also compares very well with the in vitro cAMP EC₅₀ of unlabeled RTI-13951-33 (45 nM) and is 2-fold more potent than the K_D obtained in the PPLS-HA-hGPR88-CHO membranes (85 nM). Similarly, the B_max in native tissue is 3-fold higher compared to the B_max in the PPLS-HA-hGPR88-CHO membranes (4.5 pmol/mg protein). The favorable K_D and B_max values in native tissue indicate the versatility of the [³H]RTI-33 radioligand to study GPR88 ligand-receptor interactions in native as well as recombinant systems.

Figure 4.

Saturation curve for one-site specific [³H]RTI-33 binding to wild-type mouse striatal membranes using filtration methods. Data points are the means ± SEM of three independent experiments, each performed with duplicate determinations. Inset: Scatchard plot of the same data.

In conclusion, we have developed the first radioligand for the orphan receptor GPR88 and characterized its binding properties through saturation and competition binding studies. [³H]RTI-33 can be easily prepared with a radiochemical purity of 98% and specific activity of 83.4 Ci/mmol through a 2-step procedure. [³H]RTI-33 has a high affinity for the GPR88 receptor in recombinant membrane preparations and native mouse striatal tissue. Competition binding with a selection of 2-PCCA and 2-AMPP analogs revealed that GPR88 functional agonists were able to competitively displace [³H]RTI-33. This new GPR88 radioligand represents a powerful tool to study GPR88 receptor-ligand interactions and for the discovery of novel GPR88 ligands.

Highlights.

• [³H]RTI-33 is the first radioligand for the GPR88 receptor.

• [³H]RTI-33 has high affinity for GPR88 in PPLS-HA-hGPR88-CHO membranes.

• GPR88 functional agonists competitively displace [³H]RTI-33.

Data availability

Data is available upon request.