br Surrogate ligands for GPR Although

Surrogate ligands for GPR35 Although identification of endogenously produced chemicals with agonist action at GPR35 is of considerable importance, the ligands described above are far from ideal to probe the roles of GPR35. Surrogate ligands are therefore required. Until recently, the key GPR35 agonist has been zaprinast (2-(2-propyloxyphenyl)-8-azapurin-6-one) (Table 1). Zaprinast was first identified as a GPR35 agonist by Tanaguchi et al.[22]. Like kynurenic acid, zaprinast was considerably more potent at rat than human GPR35, an observation that has also subsequently been confirmed by others 13, 23 (Figure 2). Importantly, however, zaprinast is substantially better known as an inhibitor of cGMP phosphodiesterases (PDEs), particularly PDE5 and PDE6, for which it displays low micromolar potency. If zaprinast is used as the probe, it could be difficult in many settings to disentangle the contribution of the elevation of cGMP from that resulting from the activation of GPR35. As a consequence, two groups screened the Prestwick Chemical Library® of 1120 small molecule marketed drugs and drug-like molecules for ligands able to act as agonists at human [12] or both human and rat [23] GPR35. Although both groups employed GPR35-β-arrestin 2 interaction assays, the bases of these were distinct (Box 2). Zhao et al.[12] reported two hits from the primary screen: the previously characterized ligand zaprinast and oxantel pamoate. However, in follow-up studies pamoate (4,4′-methylenebis(3-hydroxy-2-naphthoic acid)), rather than the supposed active ingredient oxantel (1-methyl-2-(3-hydroxyphenylethenyl)-1,4,5,6-tetrahydropyrimidine), was identified as the GPR35 active ligand, displaying an EC50 value of 80nM [12]. By comparison, Jenkins et al.[23] reported a wider range of hits at human GPR35 in their primary screen. These included zaprinast but also cromolyn (5,5′-(2-hydroxypropane-1,3-diyl)bis(oxy)bis(4-oxo-4H-chromene-2-carboxylic acid)) disodium, dicumarol (3,3′-methylenebis(4-hydroxy-2H-chromen-2-one)), niflumic cox pathway (2- [3-(trifluoromethyl)phenyl]amino nicotinic acid) and, importantly, both oxantel pamoate and pyrvinium (4-[(3-carboxy-2-hydroxynaphthalen-1-yl)methyl]-3-hydroxynaphthalene-2-carboxylic acid; 1-methyl-2-[(E)-2-thiophen-2-ylethenyl]-5,6-dihydro-4H-pyrimidine)) pamoate (Table 1). The presence of the supposed inactive drug congener pamoate in two separate primary screen hits alerted these researchers to the possibility that this was the common link in the responses and subsequent confirmation of pamoate as an agonist at human GPR35 with an EC50 value of 50 nM [23]. The recognition that supposedly inactive components of a mixture might have activity at a distinct target has been discussed further in light of these results [24]. Importantly, previous studies by Jenkins et al.[13] that had confirmed the species orthologue selectivity of zaprinast encouraged the authors to repeat the primary screen using rat GPR35 [23]. Although a number of hits at human GPR35 were also identified at rat GPR35, two novel hits, the closely related flavenoids luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone) and quercetin (2-(3,4-dihydroxyphenyl)- 3,5,7-trihydroxy-4H-chromen- 4-one), were now also identified. Furthermore, neither oxantel pamoate nor pyrvinium pamoate was identified when screening against rat GPR35 [23]. This suggested that pamoate might be significantly selective for human GPR35, and when pamoate was assessed in parallel at the two species orthologues, selectivity of at least 1000-fold was observed. It was clear that pamoate was not an antagonist at the rat orthologue [23] because pamoate failed to alter the potency of zaprinast to promote interactions between rat GPR35 and β-arrestin 2 [23]. This is one of the most notable examples to date of ligand selectivity at mammalian GPCR species orthologues. Interestingly, certain other hits at GPR35 (such as niflumic acid) also displayed substantial selectivity for the human orthologue [23]. This was not universal; a number of compounds were essentially equipotent at these two orthologues (including the anti-asthma medication cromolyn disodium and the vitamin K antagonist dicumarol) [23]. However, like zaprinast, luteolin displayed significant selectivity for the rat receptor [23]. A further feature of the various ligands was that they were partial agonists when compared to zaprinast as the reference compound [23]. For example, increasing concentrations of pamoate reduced human GPR35-β-arrestin 2 interactions produced by maximally effective concentrations of zaprinast [23], while the partial agonist nature of luteolin and quercetin at rat GPR35 was also demonstrated [23]. The potential selectivity of these compounds for GPR35 over other related GPCRs and potential therapeutic targets remain to be determined. This is an important issue that needs to be explored before roles for GPR35 are defined based on ex vivo or in vivo use of such ligands. This is also the case for a series of thiazolidinedione ligands originally described as GPR35 agonists in a patent from Arena Pharmaceuticals [25] and confirmed as such by Jenkins and colleagues [13]. Currently the only described antagonists of GPR35 are based on methyl-5-[(tert-butylcarbamothioylhydrazinylidene)methyl]-1-(2,4-difluorophenyl)pyrazole-4-carboxylate; that is, CID2745687 [12] (Table 1), an nM inhibitor of the human orthologue. Use of this ligand and identification of further antagonists from distinct chemical series will probably be central in defining the key functions of GPR35.