Abstract
Many members of the G protein-coupled receptor family, including examples with clear therapeutic potential, remain poorly characterised. This often reflects limited availability of suitable tool ligands with which to interrogate receptor function. In the case of GPR84, currently a target for the treatment of idiopathic pulmonary fibrosis, recent times have seen the description of novel orthosteric and allosteric agonists. Using 2-(hexylthiol)pyrimidine-4,6 diol (2-HTP) and di(5,7-difluoro-1H-indole-3-yl)methane (PSB-16671) as exemplars of each class, in cell lines transfected to express either human or mouse GPR84, both ligands acted as effective on-target activators and with high co-operativity in their interactions. This was also the case in lipopolysaccharide-activated model human and mouse immune cell lines. However in mouse bone-marrow-derived neutrophils, where expression of GPR84 is particularly high, the capacity of PSB-16671 but not of 2-HTP to promote G protein activation was predominantly off-target because it was not blocked by an antagonist of GPR84 and was preserved in neutrophils isolated from GPR84 deficient mice. These results illustrate the challenges of attempting to study and define functions of poorly characterised receptors using ligands that have been developed via medicinal chemistry programmes, but where assessed activity has been limited largely to the initially identified target.
Highlights
A substantial number of G protein-coupled receptors (GPCRs) are the targets of therapeutic medicines and have been extensively studied[1], this is not the case for many other family members
Because agonist actions of each of medium chain length fatty acids (MCFAs), embelin and 2-HTP at human GPR84 are prevented by mutation of arginine[172] of the receptor[11] that acts to co-ordinate the carboxylate of MCFAs, both embelin and 2-HTP are classified as orthosteric agonists, i.e. they bind at the same site as the MCFAs that are notionally the endogenous activators of the receptor
As with many other GPCRs, GPR84 can be activated by ligands that bind at a distinct site(s)
Summary
A substantial number of G protein-coupled receptors (GPCRs) are the targets of therapeutic medicines and have been extensively studied[1], this is not the case for many other family members. Antagonists of GPR84 are limited, with GLPG1205 and related molecules, including compound 107 (2-([1,4]dioxan-2-ylmethoxy)-9-(3-phenylamino-prop-1-ynyl)-6,7-dihydro-pyrimido[6,1-a]isoquinolin-4-one), being the only reported and studied examples[11,14] These act as high affinity, but non-competitive, blockers of the actions of both orthosteric and allosteric agonists of GPR8411 and GLPG1205 has been used to block effects of GPR84 activation in human phagocytes[14]. These conclusions are based on the inability of a GPR84 antagonist that blocked effects of PSB16671 at mouse GPR84 in both transfected cells and in the macrophage-like RAW264.7 cell line to do so in bone marrow-derived neutrophils, and because the activity of PSB-16671 is preserved in neutrophils from GPR84 knock-out mice These studies indicate the challenges and dangers of transferring outcomes from medicinal chemistry programmes into physiological studies and outcomes for poorly characterised receptors where little is known about the mode of binding of ligands that are described to have potency and affinity at that specific target. Doing so may result in incorrect assignment of functions to such a receptor
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