The GPR139 reference agonists 1a and 7c, and tryptophan and phenylalanine share a common binding site

Abstract

GPR139 is an orphan G protein-coupled receptor expressed in the brain, in particular in the habenula, hypothalamus and striatum. It has therefore been suggested that GPR139 is a possible target for metabolic disorders and Parkinson’s disease. Several surrogate agonist series have been published for GPR139. Two series published by Shi et al. and Dvorak et al. included agonists 1a and 7c respectively, with potencies in the ten-nanomolar range. Furthermore, Isberg et al. and Liu et al. have previously shown that tryptophan (Trp) and phenylalanine (Phe) can activate GPR139 in the hundred-micromolar range. In this study, we produced a mutagenesis-guided model of the GPR139 binding site to form a foundation for future structure-based ligand optimization. Receptor mutants studied in a Ca2+ assay demonstrated that residues F1093×33, H1875×43, W2416×48 and N2717×38, but not E1083×32, are highly important for the activation of GPR139 as predicted by the receptor model. The initial ligand-receptor complex was optimized through free energy perturbation simulations, generating a refined GPR139 model in agreement with experimental data. In summary, the GPR139 reference surrogate agonists 1a and 7c, and the endogenous amino acids l-Trp and l-Phe share a common binding site, as demonstrated by mutagenesis, ligand docking and free energy calculations.

Mutagenesis of GPR139 reveals ways to create gain or loss of function receptors

“…Of note is Phe109Ala, which is one of the mutations that caused a complete loss of function to all four compounds. This result is consistent with an earlier report indicating Phe109 is highly important for these ligands ability to bind and mutation to alanine resulted in a complete loss of function . Nøhr et al also indicated Asn271 as another common binding site shared by these ligands and a mutation from a hydrophilic residue Asn to a hydrophobic residue Ala also resulted in a complete loss of function.…”

“…Lastly, the larger GPR139 agonists (TC-O 9311 and JNJ-63533054), also share a less buried hydrophobic pocket defined by residues Val76, Phe79, Ile104, Val83, and Ile80. This common binding site was also previously identified by Norh et al 15 Perhaps more importantly, we were able to identify mutations that rendered the receptor either more sensitive or less sensitive to the purported amino acid ligands. This should allow for the creation of transgenic animals that will require higher or lower levels of endogenous ligand to achieve activation, and enable more insight into the in vivo function of GPR139.…”

“…An overview of hGPR139 ligand binding site with surface representation is shown in Figure . A common binding site for TC‐O 9311 and JNJ‐63533054 and endogenous amino acids L‐Trp and L‐Phe (Figure A) was previously identified by Norh et al, with a deeply buried hydrophobic region between Phe109, His187, and Trp241 (Figure B) and a polar region defined by Asn271, Arg244, and Glu108 (Figure C). The ligands were described as positioning themselves in a way that their aromatic moieties fit in the deep hydrophobic region while their polar or charged regions bind to Asn271 and Arg244.…”

“…Shehata et al also presented a combined structure‐activity relationship, and a refined pharmacophore model of GPR139 . By applying in vitro and in silico mutagenesis, they demonstrated a common binding site for GPR139 surrogate agonists, including TC‐O 9311, JNJ‐63533054, L‐Trp, and L‐Phe, indicating that residues Phe109, His187, and Asn271 are important for the binding of these ligands …”

“…14 By applying in vitro and in silico mutagenesis, they demonstrated a common binding site for GPR139 surrogate agonists, including TC-O 9311, JNJ-63533054, L-Trp, and L-Phe, indicating that residues Phe109, His187, and Asn271 are important for the binding of these ligands. 15 The goal of the present study was to further understand the critical structural motifs for GPR139 receptor activity as well as create new tools to further study the physiological role of GPR139. We used both site-directed and random mutagenesis approaches and identified series of human GPR139 mutation clones with gain-offunction, reduction-of-function as well as loss-of-function properties.…”