6-Bromo-8-(4-[³H]methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic Acid: A Powerful Tool for Studying Orphan G Protein-Coupled Receptor GPR35

Abstract

The potent and selective GPR35 agonist 6-bromo-8-(4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (12) was obtained in tritium-labeled form, designated [(3)H]PSB-13253, with a specific activity of 36 Ci (1.33 TBq)/mmol. Radiolabeling was achieved by methylation of ethyl 6-bromo-8-(4-((tert-butyldimethylsilyl)oxy)benzamido)-4-oxo-4H-chromene-2-carboxylate (19) with [(3)H]methyl tosylate followed by ester hydrolysis. The radioligand was characterized by kinetic, saturation, and competition assays at membrane preparations of Chinese hamster ovary cells recombinantly expressing the human GPR35. [(3)H]12 labeled the receptor with high affinity (KD = 5.27 nM). Binding was saturable (Bmax = 12.6 pmol/mg of protein) and reversible. Affinities of selected standard ligands and a library of amidochromen-4-one-2-carboxylates were determined. Binding data mostly correlated with potencies determined in β-arrestin assays. On the basis of the test results, several new fluorine-substituted 6-bromo-8-benzamidochromen-4-one-2-carboxylic acids were obtained, which represent the most potent GPR35 agonists known to date. 6-Bromo-8-(2,6-difluoro-4-methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (83; Ki = 0.589 nM, EC50 = 5.54 nM) showed the highest affinity with a Ki value in the subnanomolar range.

GPR35 mediates lodoxamide‐induced migration inhibitory response but not CXCL17‐induced migration stimulatory response in THP‐1 cells; is GPR35 a receptor for CXCL17?

“…Several endogenous molecules, such as lysophosphatidic acid, kynurenic acid , cGMP and reverse T3, were proposed as ligands (Wang et al, ; Oka et al, ; Jenkins et al, ; Deng et al, ; Southern et al, ), but their potencies were too low (in the μM range) to support their roles as GPR35 ligands (Divorty et al, ). Synthetic surrogate agonists, such as zaprinast , pamoic acid , compound 1, PSB‐13253 and lodoxamide, have been successfully identified or developed (Taniguchi et al, ; Jenkins et al, ; Zhao et al, ; Funke et al, ; Neetoo‐Isseljee et al, ; Thimm et al, ; MacKenzie et al, ), and of these lodoxamide most potently interacts with human and rodent GPR35 (MacKenzie et al, ). Selective synthetic antagonists for GPR35 were also developed, that is, CID‐2745687 (also known as SPB05142 and ML194) and CID‐2786812 (ML145) (Zhao et al, ; Heynen‐Genel et al, ,b; Jenkins et al, ).…”

Identification of Novel G Protein–Coupled Receptor 143 Ligands as Pharmacologic Tools for Investigating X-Linked Ocular Albinism

“…A widely applied assay for library screening is the β-arrestin recruitment assay, because many ligands also show (biased) agonism in this assay. 22–26 The assay is well suited for orphan GPCRs, because a positive control is not essential. 27,28 GPR143 colocalizes with β-arrestin 19 ; therefore, we hypothesized that the β-arrestin recruitment assay was suitable for high-throughput screening.…”

Development of [3H]2-Carboxy-4,6-dichloro-1H-indole-3-propionic Acid ([3H]PSB-12150): A Useful Tool for Studying GPR17

“…20 In contrast, Mg 2+ ions may enhance the formation of a high-affinity agonist−receptor−G protein complex acting as a positive allosteric modulator, and the same effect has recently been observed with an agonist radioligand at GPR35. 21 Thus, 50 mM TRIS buffer, pH 7.4, containing 10 mM MgCl 2 was found to be a suitable buffer for performing radioligand binding studies with [ 3 H]PSB-12150, and it was therefore used in all further experiments.…”