Impact of species variability and 'probe-dependence' on the detection and in vivo validation of allosteric modulation at the M4 muscarinic acetylcholine receptor

Abstract

Background and purpose: We recently characterized LY2033298 as a novel allosteric modulator and agonist at M(4) muscarinic acetylcholine receptors (mAChRs). Evidence also suggested a difference in the potency of LY2033298 at rodent relative to human M(4) 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 approach: LY2033298 was investigated in vitro in CHO cells stably expressing human or mouse M(4) mAChRs, using assays of agonist-induced ERK1/2 or GSK-3α phosphorylation, [(35) S]-GTPγS binding, or effects on equilibrium binding of [(3) H]-NMS and ACh. The in vivo actions of LY2033298 were investigated in a mouse model of amphetamine-induced locomotor activity. The function of LY2033298 was examined in combination with ACh, oxotremorine or xanomeline.

Key results: LY2033298 had similar affinities for the human and mouse M(4) mAChRs. However, LY2033298 had a lower positive co-operativity with ACh at the mouse relative to the human M(4) mAChR. At the mouse M(4) 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 M(4) mAChR necessitated the co-administration of LY2033298 with oxotremorine in order to show in vivo efficacy of LY2033298.

Conclusions and implications: These results provide evidence for species variability when comparing the allosteric interaction between LY2033298 and ACh at the M(4) 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.

Figure 4

Effects of LY2033298 on the ability of ACh or oxotremorine to promote the phosphorylation of GSK-3α at the (A) human or (B) mouse M₄ mAChR stably transfected in CHO cells. Data represent the mean of five experiments performed in duplicate.

Figure 1

Effects of ACh or LY2033298 on ERK1/2 phosphorylation mediated by (A) human or (B) mouse M₄ mAChRs stably transfected in CHO cells. Data points represent the mean ± SEM obtained from four to five experiments performed in duplicate.

Figure 2

Effects of LY2033298 on the ability of ACh to (A) compete with the binding of the orthosteric antagonist, [³H]-NMS in membranes stably expressing the human (solid symbols) or mouse M₄ (open symbols) mAChR, (B) promote the phosphorylation of ERK1/2 at the human M₄ mAChR stably transfected in CHO cells, and (C) promote the phosphorylation of ERK1/2 at the mouse M₄ mAChR stably transfected in CHO cells. Data points represent the mean ± SEM obtained from (A) five, (B) four or (C) five experiments performed in duplicate.

Figure 3

Effects of LY2033298 on the ability of (A) oxotremorine or (B) xanomeline to promote the phosphorylation of ERK1/2 at the mouse M₄ mAChR stably transfected in CHO cells. Data represent the mean of (A) five or (B) four experiments performed in duplicate. (C) Nonlinear regression of the pEC₅₀ values determined for ACh-, oxotremorine- or xanomeline-mediated ERK1/2 at the mouse M₄ mAChR, or for ACh at the human M₄ mAChR, in the absence or presence of LY2033298 according to an allosteric ternary complex model (Equation 4).

Figure 5

Effects of LY2033298 on the ability of (A) ACh (B) oxotremorine or (C) xanomeline, to promote the binding of [³⁵S]-GTPγS to activated Gα proteins via the human or mouse M₄ mAChR stably expressed in CHO cell membranes. Data represent the mean of three experiments performed in triplicate.

Figure 6

A. The effect of amphetamine alone and in combination with either haloperidol, clozapine, oxotremorine or xanomeline on locomotor activity measured as counts. Asterisks indicate significant differences from control untreated animals, P < 0.05, post hoc Dunnett's test. B. The effects of LY2033298 (10 mg·kg⁻¹) in combination with amphetamine, amphetamine and oxtremorine, or amphetamine and xanomeline. Asterisks indicate significant differences from control untreated animals, P < 0.05, post hoc Dunnett's test. For clarity of comparisons locomotor activity in control untreated animals and amphetamine alone are included in the figure.