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037P University of Leicester, UK
6th Focused Meeting on Cell Signalling

 

 

Pharmacological Evaluation of CNO as a Tool to Determine in vivo GPCR Function

We have developed a chemical genetic approach to study the function of the M1 muscarinic receptor in vivo. This method involves mutating the receptor to remove receptor responsiveness to its natural ligand, acetylcholine (ACh), while simultaneously engender activity to an otherwise inert compound, Clozapine-N-oxide (CNO). These mutations, Y3.33C and A5.46G, were initially developed within the human M3 muscarinic receptor (1) and are conserved residues located within the binding pocket of members of the muscarinic receptor family. Prior to the generation of a knock-in mouse model expressing the mutant receptor (termed M1 RASSL) we have characterised the pharmacological properties of CNO in vitro.

In competition radioligand binding studies at the human M1 receptor using 3H-NMS, CNO competitively bound to the orthosteric site, producing a Kd of -5.35 ± 0.04, compared to ACh binding with a Kd of -4.85 ± 0.07 Using an inositol phosphate accumulation assay, figure 1, we show that CNO was able to cause inositol phosphate accumulation with EC50 of -5.61 ± 0.23 and -5.29 ± 0.27 at the human and mouse wild-type M1 muscarinic receptors, respectively. The compound produced maximal response ~30% of the response produced by ACh and hence behaved as a partial agonist. Functional antagonism of the mouse and human M1 receptors using inositol phosphate accumulation show CNO functionally antagonise the ACh response at the mouse M1, with a pA2 of -5.35 ± 0.09, when the two ligands were co-added.


Figure 1. Inositol phosphate accumulation concentration response curves for human M1-WT (a) and mouse M1-WT (b) receptors with ACh and CNO. Data are shown as mean ± SEM n=3.

These data suggest that CNO, unlike previously thought, possesses pharmacological activities at the M1 muscarinic receptors. It is therefore important to select the right dosage for in vivo studies in order to avoid toxic or unwanted off target effects. To further evaluate CNO we will characterise its pharmacological properties at other members of the muscarinic receptor family, as any interactions between CNO and these receptors will also have an effect on the in vivo dosing.

Armbruster BN et al. (2007) PNAS 104: 5163–5168.