Probe Dependence in the Allosteric Modulation of a G Protein-Coupled Receptor: Implications for Detection and Validation of Allosteric Ligand Effects

Abstract

We recently described 3-amino-5-chloro-6-methoxy-4-methylthieno [2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298) as a novel allosteric modulator of M 4 muscarinic acetylcholine (ACh) receptors (mAChRs) on the basis of its ability to preferentially potentiate the actions of ACh at the M 4 mAChR subtype. In the current study, we show that LY2033298 can also bind to the M 2 mAChR and mediate robust positive or negative allosteric effects, depending on the orthosteric ligand used as a probe of receptor activity. This finding of striking "probe dependence" indicates that the previously described selectivity of the modulator does not arise as a consequence of selective affinity for a poorly conserved allosteric site but rather is due to subtype-selective cooperativity with ACh upon interaction with a common allosteric binding site. Moreover, a comparison of the effects of the modulator on orthosteric ligand affinity relative to signaling through a [ 35 S]guanosine 5Ј-O-(3-thio)triphosphate or extracellular signalregulated kinase 1/2 phosphorylation assay at the M 2 mAChR revealed that, although the effects on binding were positive in all instances, the effects on signaling were either positive or strongly negative, depending on the agonist and the pathway. Mutational analysis identified residues Tyr177 and Trp99 3.28 (Ballesteros and Weinstein numbers are provided in superscript to indicate relative position of residues within the transmembrane domain) as contributing to the binding of LY2033298, whereas the orthosteric site residues, Tyr104 3.33 and Tyr403 6.51 , contributed to the ability of the ligand to impose pathway-biased modulation. Taken together, these findings have important implications for the detection and validation of allosteric modulators of G protein-coupled receptors (GPCRs), because they highlight the potential for ligand misclassification or lack of appreciation of off-target allosteric activities.

Allosteric Modulation of Endogenous Metabolites as an Avenue for Drug Discovery

“…To validate our hypothesis that metabolites of endogenous ligands can be allosterically modulated at the GPCR of the parental ligand, we performed an initial screen using a representative allosteric ligand for three different model systems: the M 2 mAChR, the A 1 -AR, and the GLP-1R. In a recent study, we characterized LY2033298 as an allosteric modulator of the M 2 mAChR (Valant et al, 2012). PD81723 is a well accepted allosteric modulator of the A 1 -AR (Bruns and Fergus, 1990), and we have also recently identified a series of low-molecular-weight pyrimidine-based compounds that activate the GLP-1R allosterically, the most potent representative being BETP (designated compound B in Sloop et al, 2010).…”

“…[ 35 S]GTP␥S binding was determined as described previously (Valant et al, 2012). M 2 mAChR FlpInCHO cell membranes (5-25 g) were equilibrated in a 500-l total volume of assay buffer containing 10 M guanosine 5Ј-diphosphate and a range of concentrations of ligands (ACh or Ch) in the absence or presence of LY2033298 (0.1-10 M) at 30°C for 60 min.…”

“…In this study, we investigated the potential to allosterically modulate the activity of the predominant, inactive metabolite of the physiological ligand at three different GPCRs for which small molecule allosteric modulators have been described: the GLP-1R (Knudsen et al, 2007;Koole et al, 2010;Sloop et al, 2010), the M 2 muscarinic acetylcholine receptor (M 2 mAChR) (Valant et al, 2012), and the adenosine A 1 receptor (A 1 -AR) (Bruns and Fergus, 1990) (Supplemental Fig. 1).…”

Development of M1 mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

“…Another consideration with regard to screening is the nature of the receptor, be it rat, human recombinant, or human native. As previously highlighted with LY2033298 (10) 76,78 and Ndesmethylclozapine (35), 128 identification of species-dependent variability in both cooperativity and mode of action can lead to the discovery of altered selectivity and functional profiles that may have had deleterious results in a clinical setting if not identified earlier. Furthermore, the potential for allosteric ligands to elicit different physiological and toxicological outcomes in animal and human studies poses considerable challenges to the traditional drug discovery pipeline that will be difficult to resolve.…”

The Therapeutic Potential of Allosteric Ligands for Free Fatty Acid Sensitive GPCRs

“…Complicating the issue, if the desired compound is an allosteric modulator (either PAM or NAM) the screening must be conducted in the presence of an orthosteric agonist. Since it is now recognised that allosteric modulators do not necessary produce the same effect on all orthosteric agonists, a phenomena described as 'probe dependence' [25,56], the particular orthosteric ligand chosen to use in the screen is critical, though in most cases the endogenous ligand itself is the obvious choice. If instead the goal is to identify allosteric agonists, it is important to confirm any identified 'hits' are actually allosteric in nature through follow-up studies.…”