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  • br Introduction Our understanding of how ligands interact


    Introduction Our understanding of how ligands interact with G protein coupled receptors is evolving, particularly the recognition that some have the ability to preferentially activate a subset of intracellular signalling cascades – so called pathway biased ligands [1]. Additionally, it is now accepted that recruitment of β-arrestin that occurs following activation of the majority of GPCRs not only results in receptor desensitisation and subsequent internalisation but may also contribute to cellular responses involved in normal physiology and disease such as cell migration and proliferation [2]. opioid receptor Therefore, exploiting ligand bias is likely to lead to the development of more effective and better tolerated medicines. This has so far been most clearly demonstrated for the μ opioid receptor where the agonist TRV130, a molecule that discriminates between beneficial analgesia and detrimental adverse effects such as respiratory depression and nausea, exhibited an improved therapeutic profile compared to morphine in a randomized, double-blind, placebo-controlled, crossover study in healthy volunteers [3]. Whereas bias has been considered a property of synthetic ligands it has recently been reported that for example endogenous opioids also show bias at the μ-opioid receptor [4] indicating that the presence of multiple ligands for a receptor, rather than simply representing physiological redundancy, may allow for nuanced cell specific signalling. Distinct roles for the three endogenous endothelin (ET) peptides are emerging in development and in, for example, ovarian physiology but whether pathway bias may contribute to the physiology and pathophysiology of the endogenous peptides in the ET system has not been explored. In opioid receptor the potential for targeting the endothelin receptors with synthetic biased ligands is starting to be considered. This brief review discusses current research on biased signalling at the ET receptors and therapeutic areas of interest.
    ET receptors and probe dependence Some of the pharmacology of the endothelin receptors has over the last 20years been described as atypical; not conforming to the basic tenets of receptor pharmacology. Particularly, this has been in differences in the behaviour of the endogenous peptides and synthetic agonists with respect to reversal by washout or blockade/reversal of responses by antagonists in in vitro studies [5], [6]. It is now apparent that for a particular receptor multiple active conformations, rather than just one, are possible and ligands can stabilize different conformations of a receptor that may activate subsets of available down-stream pathways. Therefore, some of the atypical pharmacology reported for ET receptors may be consistent with these agonists showing a degree of functional selectivity, although differences in ligand-receptor kinetics may also contribute to these observations. Additionally, because of the allosteric nature of the interaction of ligand–GPCR–intracellular protein (e.g. G protein) affinity measured in binding assays may differ from affinity measured in functional assays, specifically if different agonists stabilize particular receptor conformations then this allows the potential for orthosteric antagonists to demonstrate agonist specific functional affinities – consistent with previously reported atypical pharmacology of probe dependence [7].
    There has been at least one report in vitro that ET peptides exhibit bias at the ETA receptor with ET-1 and ET-2 suggested to elicit their long lasting constrictor responses via different mechanisms that was also vascular bed dependent [8]. We have previously published data on both the potency and efficacy of endothelin peptides and sarafotoxins as constrictors of human saphenous vein [9] and in β-arrestin recruitment assays [10]. These data highlighted differences in the relative potencies and efficacies of these agonists in the ETA mediated constrictor and β-arrestin recruitment assays indicative of bias. Several methods for determining pathway bias from such data have been reported including determination of transducer coefficients τ/KA, as described by van Westhuizen and colleagues [11]. We have applied this method to our existing data to determine whether the endogenous ET peptides and related sarafotoxin 6b (S6b) show any evidence of bias in the G protein dependent vasoconstrictor assay and G protein independent β-arrestin recruitment assay. Determining bias requires designation of a reference compound that is preferably the endogenous ligand. For the cardiovascular ETA receptor the most appropriate reference ligand is ET-1. All data are expressed as a % of the maximum ET-1 response and analysed as described [10], to obtain values of log10(τ/KA) that are used for subsequent determination of bias factors. Fig. 1 shows that whilst ET-1, ET-2 and S6b are full agonists in the constrictor assay (the ET-3 curve is incomplete at the maximum possible bath concentration) ET-3 and S6b are both partial agonists in the β-arrestin assay.