The selectivity of Duo3 for the M1-M4 muscarinic receptor subtypes

Asma Baig, *Christian Tränkle, †Ulrike Holzgrabe & Nigel J.M. Birdsall, MRC National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK, *Dept. Pharmacology & Toxicology, University of Bonn, FRG, †Institute of Pharmacy, University of Würzburg, FRG.

Muscarinic acetylcholine receptors have two allosteric sites (Lazareno et al., 2002). One site binds ligands such as gallamine and brucine (‘gallamine’ site) and the other binds WIN 62,577, WIN 51,708 and staurosporine (‘WIN’ site). Certain ‘atypical’ allosteric ligands, such as Duo3 (4,4´-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1´-propane-1,3-diyl-bis-pyridinium dibromide), are characterised by their steep slope factors (>1) in their allosteric kinetic actions at the M2 receptor subtype but the mechanism that gives rise to these steep slopes is not understood.

Membranes from CHO cells expressing the M1-M4 subtypes (1-5 pmoles/mg protein) were prepared as described previously (Lazareno et al., 2002). Radioligand binding assays were carried out at 30o in 5mM Na/K phosphate pH 7.4 or in 100mM NaCl/10mM MgCl2/0.2mM GTP/20mM Hepes, pH 7.4. The equilibrium and dissociation assays to determine the affinities, cooperativities and slope factors of Duo3 using the antagonist [3H]N-methylscopolamine ([3H] NMS) have been described in detail. Data are expressed as means ± sem (n).

In the low ionic strength buffer, the dissociation experiments gave log potencies and slope factors for Duo3 at the NMS-occupied receptors of (6.64 ± 0.04, 1.32 ± 0.03, 4; 7.24 ± 0.05, 1.25 ± 0.05, 9; 6.47 ± 0.06, 1.09 ± 0.10, 5; 6.86 ± 0.03, 1.24 ± 0.08, 3) at M1-M4 receptors respectively, with the slope factors being significantly greater than 1 at M1, M2 and M4 receptors (P < 0.05). In this buffer, equilibrium experiments at M2 receptors could be analysed by the allosteric ternary complex model to give a log affinity for Duo3 at the unoccupied receptor of 7.95 ± 0.05 (5) and a cooperativity with NMS of 0.07 ± 0.01 (5). Because of the higher negative cooperativity between NMS and Duo3 at the other subtypes, those inhibition curves could not be distinguished from simple competition curves (pKi values,M1 8.58 ± 0.07, 6; M3 7.73 ± 0.07, 3; M4 7.92 ± 0.13, 4) with, surprisingly, the slope factors being very close to, and not significantly different from, 1 (1.01 ± 0.06, 1.07 ± 0.03; 1.02 ± 0.03, respectively P > 0.05). In the higher ionic strength buffer, the Duo3/ [3H]NMS inhibition curves could not be distinguished from simple competition curves at all subtypes (pKi values, 7.97 ± 0.03, 6; 8.19 ± 0.04, 6; 7.35 ± 0.03, 2; 7.81 ± 0.01, 4) with the slope factors again being very close to, and not significantly different from, 1 (1.07 ± 0.07, 1.02 ± 0.05; 1.06 ± 0.06; 1.09 ± 0.07, respectively P > 0.05). In this buffer, the ca. 20–40 fold lower potencies of Duo3 in the kinetic assays precluded accurate quantitation of these data.

The high affinity of Duo3 and its unanticipated M1 potency make it a possible candidate for an allosteric radioligand at this subtype. Duo3 only appears to exhibit its ‘atypical’ behaviour at the [3H]NMS-occupied receptor, as monitored by the [3H]NMS kinetic assays, and not when it binds to the unoccupied receptor.

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