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We have previously reported that in rat hippocampal area CA1, the A1R-selective agonist, BnOCPA, potently inhibited excitatory synaptic transmission but did not cause membrane hyperpolarisation in CA1 pyramidal neurons, as would be expected of A1R agonists. This functional discrimination by BnOCPA may arise from its ability, in cAMP inhibition assays, to selectively activate only Gob out of the six Gαi/o subtypes. This may explain why BnOCPA is a potent analgesic that does not cause sedation or cardiorespiratory depression in the rat. Since many preclinical studies are performed using mice, we have here investigated whether BnOCPA’s functional discrimination extends to the mouse. While the potency of BnOCPA against the inhibition of hippocampal synaptic transmission was comparable between rats and mice, we discovered that low concentrations of BnOCPA hyperpolarised mouse CA1 neurons and reduced both their input resistance and firing rate in an A1R-dependent manner. In interleaved experiments we confirmed our previous observations in the rat that concentrations of BnOCPA equivalent to those tested in the mouse had little or no effect on membrane potential or input resistance. Using NanoBRET binding we established that BnOCPA had similar affinity at the mouse and rat A1Rs, and displayed little difference in G protein coupling, as determined using the TRUPATH assay. Thus, although the mechanism for the loss of BnOCPA functional selectivity between pre- and postsynaptic receptors in the mouse hippocampus is currently unclear, it may stem from differences in expression of the individual G proteins subunits or the coupling to murine K+ channels.

One third of all medicines act at G protein-coupled receptors (GPCRs). However, the identification of new therapeutic GPCR targets is limited by a fundamental property of GPCRs: their propensity to couple to different G protein alpha subunits (Gα) leading to multiple downstream cellular effects. This is especially the case for adenosine A 1 receptors (A 1 Rs), the activation of which results in unwanted cardiorespiratory effects, severely limiting their clinical potential. We have discovered that the novel A 1 R agonist, BnOCPA, unlike typical A 1 R agonists, has a unique and highly selective Gα activation preference. Moreover, we found that BnOCPA is a potent and powerful analgesic without causing bradycardia, hypotension or respiratory depression. BnOCPA thus demonstrates a hitherto unknown Gα-selective activation of the native A 1 R, sheds new light on the fundamentals of GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of biased agonism. Abbreviated summary We describe the selective activation of an adenosine A 1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of biased agonism.

SummaryThe development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, BnOCPA, is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPA’s unique and exquisitely selective activation of Gob among the six Gαi/o subtypes, and in the absence of β-arrestin recruitment. BnOCPA thus demonstrates a highly-specific Gα-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective Gα agonism.Short summaryWe describe the selective activation of an adenosine A1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of sedation or cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of selective Gα agonism.