Impact of species variability and ‘probe‐dependence’ on the detection andin vivovalidation of allosteric modulation at the M₄muscarinic acetylcholine receptor

Abstract

We recently characterized LY2033298 as a novel allosteric modulator and agonist at M4 muscarinic acetylcholine receptors (mAChRs). Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M4 mAChRs. The current study investigated the basis for the species difference of this modulator and used this knowledge to rationalize its in vivo actions. EXPERIMENTAL APPROACHLY2033298 was investigated in vitro in CHO cells stably expressing human or mouse M4 mAChRs, using assays of agonistinduced ERK1/2 or GSK-3a phosphorylation, [ KEY RESULTSLY2033298 had similar affinities for the human and mouse M4 mAChRs. However, LY2033298 had a lower positive co-operativity with ACh at the mouse relative to the human M4 mAChR. At the mouse M4 mAChR, LY2033298 showed higher co-operativity with oxotremorine than with ACh or xanomeline. The different degrees of co-operativity between LY2033298 and each agonist at the mouse relative to the human M4 mAChR necessitated the co-administration of LY2033298 with oxotremorine in order to show in vivo efficacy of LY2033298. CONCLUSIONS AND IMPLICATIONSThese results provide evidence for species variability when comparing the allosteric interaction between LY2033298 and ACh at the M4 mAChR, and also highlight how the interaction between LY2033298 and different orthosteric ligands is subject to 'probe dependence'. This has implications for the validation of allosteric modulator actions in vivo.

Antipsychotic Drug-Like Effects of the Selective M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator VU0152100

“…Our data with VU0152100 also validate previously reported effects using the structurally distinct M 4 PAM LY2033298 when given in combination with a sub-threshold dose of the non-selective mAChR agonist oxotremorine (Chan et al, 2008;Leach et al, 2010;Suratman et al, 2011). However, LY2033298 does not provide an optimal tool compound for rodent studies in that it has relatively low potency at the rat M 4 mAChR (Chan et al, 2008;Leach et al, 2010) and displays only weak cooperativity with ACh, the endogenous agonist of M 4 (Suratman et al, 2011), but has high cooperativity with the synthetic mAChR agonist oxotremorine.…”

“…However, LY2033298 does not provide an optimal tool compound for rodent studies in that it has relatively low potency at the rat M 4 mAChR (Chan et al, 2008;Leach et al, 2010) and displays only weak cooperativity with ACh, the endogenous agonist of M 4 (Suratman et al, 2011), but has high cooperativity with the synthetic mAChR agonist oxotremorine. As LY2033298 induces robust potentiation of oxotremorine, but not ACh effects on M 4 , it has no behavioral effects when administered alone unless coadministration with oxotremorine (Suratman et al, 2011). Our finding that VU0152100 has robust efficacy in potentiating ACh responses in vitro and in multiple in vivo models when administered alone supports this interpretation.…”

“…These studies provided important data supporting the potential for the development of M 4 PAMs for the treatment of psychosis. Conversely, LY2033298, another M 4 PAM, had no effect alone in preclinical models, but did potentiate the effects of a sub-threshold dose of the non-selective mAChR agonist oxotremorine (Chan et al, 2008;Leach et al, 2010, Suratman et al, 2011. The lack of efficacy observed with LY2033298alone when used in vivo was likely due to the lower potency of this M 4 PAM at the rat M 4 mAChR and its low cooperativity with the endogenous agonist ACh at the rodent receptor (Suratman et al, 2011).…”

“…Conversely, LY2033298, another M 4 PAM, had no effect alone in preclinical models, but did potentiate the effects of a sub-threshold dose of the non-selective mAChR agonist oxotremorine (Chan et al, 2008;Leach et al, 2010, Suratman et al, 2011. The lack of efficacy observed with LY2033298alone when used in vivo was likely due to the lower potency of this M 4 PAM at the rat M 4 mAChR and its low cooperativity with the endogenous agonist ACh at the rodent receptor (Suratman et al, 2011). However, these studies also raise a potential concern that there may not be sufficient endogenous cholinergic tone to observe robust activity in key limbic and cortical regions when an M 4 PAM is administered alone.…”

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.…”