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. 2012 Dec;343(3):683-95.
doi: 10.1124/jpet.112.198945. Epub 2012 Sep 11.

Antagonists of GPR35 display high species ortholog selectivity and varying modes of action

Laura Jenkins  1 , Nicholas Harries, Jennifer E Lappin, Amanda E MacKenzie, Zaynab Neetoo-Isseljee, Craig Southern, Edward G McIver, Stuart A Nicklin, Debra L Taylor, Graeme Milligan
Affiliations

Antagonists of GPR35 display high species ortholog selectivity and varying modes of action

Laura Jenkins et al. J Pharmacol Exp Ther. 2012 Dec.

Abstract

Variation in pharmacology and function of ligands at species orthologs can be a confounding feature in understanding the biology and role of poorly characterized receptors. Substantial selectivity in potency of a number of GPR35 agonists has previously been demonstrated between human and rat orthologs of this G protein-coupled receptor. Via a bioluminescence resonance energy transfer-based assay of induced interactions between GPR35 and β-arrestin-2, addition of the mouse ortholog to such studies indicated that, as for the rat ortholog, murine GPR35 displayed very low potency for pamoate, whereas potency for the reference GPR35 agonist zaprinast was intermediate between the rat and human orthologs. This pattern was replicated in receptor internalization and G protein activation assays. The effectiveness and mode of action of two recently reported GPR35 antagonists, methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate (CID-2745687) and 2-hydroxy-4-[4-(5Z)-5-[(E)-2-methyl-3-phenylprop-2-enylidene]-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]butanoylamino)benzoic acid (ML-145), were investigated. Both CID-2745687 and ML-145 competitively inhibited the effects at human GPR35 of cromolyn disodium and zaprinast, two agonists that share an overlapping binding site. By contrast, although ML-145 also competitively antagonized the effects of pamoate, CID-2745687 acted in a noncompetitive fashion. Neither ML-145 nor CID-2745687 was able to effectively antagonize the agonist effects of either zaprinast or cromolyn disodium at either rodent ortholog of GPR35. These studies demonstrate that marked species selectivity of ligands at GPR35 is not restricted to agonists and considerable care is required to select appropriate ligands to explore the function of GPR35 in nonhuman cells and tissues.

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Figures

Fig. 1.
Fig. 1.
Structures of the three agonist ligands, zaprinast, cromolyn disodium, and pamoate, and the two previously reported antagonists, CID-2745687 and ML-145, are shown.
Fig. 2.
Fig. 2.
Pamoate is a GPR35 partial agonist with high selectivity for the human ortholog. A to C, BRET-based GPR35-β-arrestin-2 interaction assays were performed in HEK293T cells using human (●), mouse (■), or rat (▴) FLAG-GPR35-eYFP constructs. The effects of varying concentrations of zaprinast (A), cromolyn disodium (B), and pamoate (C) were assessed. D, the potency and relative efficacy of these ligands at human GPR35 is displayed. Data are representative of at least six different experiments.
Fig. 3.
Fig. 3.
Zaprinast promotes internalization of both human and rat GPR35-eYFP. Flp-In TREx 293 cells able to express either human (top) or rat (bottom) FLAG-GPR35-eYFP upon addition of doxycycline were induced with this antibiotic for 24 h. Such cells were then exposed to vehicle (left) or zaprinast (1 × 10−5 M) (right) for 30 min before imaging.
Fig. 4.
Fig. 4.
Pamoate-mediated internalization of GPR35 is restricted to the human ortholog. A to C, Flp-In TREx 293 cells able to express human (●), mouse (■), or rat (▴) FLAG-GPR35-eYFP constructs upon addition of doxycycline were induced with this antibiotic for 24 h. The capacity of varying concentrations of zaprinast (A), cromolyn disodium (B), or pamoate (C) to promote internalization of the receptor constructs was then assessed and quantified by using an ArrayScan high content imager. D, the potency and relative efficacy of these ligands at human GPR35 is displayed. Data are representative of at least six different experiments.
Fig. 5.
Fig. 5.
CID-2745687 is a potent antagonist in β-arrestin-2 interaction assays only at human GPR35. A to C, BRET-based GPR35-β-arrestin-2 interaction assays as in Fig. 2 were performed by using human (●), mouse (■), or rat (▴) FLAG-GPR35-eYFP. In each case an EC80 concentration of zaprinast (A), cromolyn disodium (B), or pamoate (only at human GPR35) (C) was added along with varying concentrations of CID-2745687.
Fig. 6.
Fig. 6.
ML-145 is also a highly selective human GPR35 antagonist in β-arrestin-2 interaction assays. A to C, experiments were conducted as in Fig. 5 except that ML-145 replaced CID-2745687. A, zaprinast. B, cromolyn disodium. C, pamoate.
Fig. 7.
Fig. 7.
CID-2745687 and ML-145 both block agonist-mediated internalization of human but not rodent forms of GPR35. A to C, the ability of varying concentrations of zaprinast (A), cromolyn disodium (B), and pamoate (C) to promote internalization of human GPR35-eYFP and the capacity of either CID-2745687 (○) or ML-145 (▴) (1 × 10 −5 M) to prevent this was assessed via high content imaging using an ArrayScan high content imager. D and E, in equivalent studies the capacity of varying concentrations of zaprinast to cause internalization of rat (D) and mouse (E) GPR35-eYFP and any capacity of CID-2745687 and ML-145 (1 × 10−5 M) to prevent this was assessed.
Fig. 8.
Fig. 8.
CID-2745687 and ML-145 block agonist-mediated internalization of human GPR35 in a concentration-dependent fashion. Experiments akin to those of Fig. 7 examined the ability of varying concentrations of ML-145 (A and C) or CID-2745687 (B and D) to prevent internalization of human (A and B) or mouse (C and D) GPR35 promoted by an EC80 concentration of the identified agonist ligands.
Fig. 9.
Fig. 9.
GPR35 stimulates IP1 accumulation when coexpressed with suitable chimeric G protein α subunits. A, HEK293T cells were transfected with or without human FLAG-GPR35-eYFP alongside either Gqi5 or Gq135 Gα subunits. Basal (open bars) IP1 accumulation and effects of zaprinast (1 × 10 −5 M) on IP1 accumulation (filled bars) were measured. A positive control was provided after transfection of the M3 muscarinic acetylcholine receptor and addition of the agonist carbachol (1 × 10 −3 M). B, after cotransfection of human FLAG-GPR35-eYFP with either Gqi5 (●) or Gq135 (■) Gα subunits the effect of various concentrations of zaprinast were assessed. C, after transfection of human (●), mouse (■), or rat (▴) FLAG-GPR35-eYFP with Gq135 the ability of varying concentrations of zaprinast (left), pamoate (center), or cromolyn disodium (right) to promote IP1 accumulation was assessed.
Fig. 10.
Fig. 10.
CID-2745687 and ML-145 block both constitutive and agonist-mediated IP1 accumulation only at the human ortholog of GPR35. The capacity of varying concentrations of ML-145 (A) or CID-2745687 (B) to limit IP1 accumulation in response to EC80 concentrations of zaprinast (left), pamoate (center), or cromolyn (right) was assessed in cells transfected to express the noted ortholog of FLAG-GPR35-eYFP with Gq135. Note the marked reduction of IP1 below basal levels (defined as 0% on y-axis), which is indicative of inverse agonism, produced by higher concentrations of ML-145 (A) and CID-2745687 (B) at the human ortholog.
Fig. 11.
Fig. 11.
ML-145 is a competitive antagonist at human GPR35, whereas the nature of antagonism by CID-2745687 depends on the identity of the agonist. BRET-based GPR35-β-arrestin-2 interaction assays were performed in HEK293T cells as in Fig. 2 by using human FLAG-GPR35-eYFP. The effects of varying concentrations of ML-145 (A-C) or CID-2745687 (D-F) on concentration-response curves to zaprinast (A and D), cromolyn disodium (B and E), and pamoate (C and F) are shown in representative experiments.
Fig. 12.
Fig. 12.
Zaprinast and cromolyn disodium probably share a common binding site on human GPR35. BRET-based GPR35-β-arrestin-2 interaction assays were performed in HEK293T cells as in Fig. 11 by using human FLAG-GPR35-eYFP. Concentration-response curves to zaprinast were performed in the presence of a range of submaximally effective concentrations of cromolyn disodium. The EC50 of zaprinast was unaffected by the copresence of cromolyn disodium.
Fig. 13.
Fig. 13.
Sequence alignment of human, mouse, and rat orthologs of GPR35. The sequence of the short isoform of human GPR35 is aligned with the mouse and rat orthologs. Asterisks indicate amino acid identity across these orthologs, and colons indicate conservative and full-stop semiconservative substitutions between species. Potential transmembrane domains are boxed. The DRY domain and the NPXXY region (light shading), two defining sequence elements of rhodopsin-like, class A GPCRs are highlighted, as are arginine (R) 3.36 and tyrosine (Y) 3.32 (dark shading). Mutation of either of these residues to alanine (Jenkins et al., 2011) eliminates the function of all GPR35 agonists for which this has been assessed (MacKenzie et al., 2011). As such, these residues probably contribute to the ligand binding pocket.
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