We studied the interactions of strychnine, brucine, and three of the N-substituted analogues of brucine with [3 H]N-methylscopolamine (NMS) and unlabeled acetylcholine at m1-m5 muscarinic receptors using equilibrium and nonequilibrium radioligand binding studies. The results were consistent with a ternary allosteric model in which both the primary and allosteric ligands bind simultaneously to the receptor and modify the affinities of each other. The compounds had K d values in the submillimolar range, inhibited [ 3 H]NMS dissociation, and showed various patterns of positive, neutral, and negative cooperativity with [ 3 H]NMS and acetylcholine, but there was no predictive relationship between the effects. Acetylcholine affinity was increased ϳ2-fold by brucine at m1 receptors, ϳ3-fold by Nchloromethyl brucine at m3 receptors, and ϳ1.5-fold by brucine-N-oxide at m4 receptors. The existence of neutral cooperativity, in which the compound bound to the receptor but did not modify the affinity of acetylcholine, provides the opportunity for a novel form of drug selectivity that we refer to as absolute subtype selectivity: an agent showing positive or negative cooperativity with the endogenous ligand at one receptor subtype and neutral cooperativity at the other subtypes would exert functional effects at only the one subtype, regardless of the concentration of agent or its affinities for the subtypes. Our results demonstrate the potential for developing allosteric enhancers of acetylcholine affinity at individual subtypes of muscarinic receptor and suggest that minor modification of a compound showing positive, neutral, or low negative cooperativity with acetylcholine may yield compounds with various patterns of cooperativity across the receptor subtypes.

WIN 51,pyrimido [1,2-a] 3 H]NMS dissociation from M 3 receptors indicate that PG987 binds reversibly to a site distinct from that to which gallamine and strychnine bind: in contrast, PG987 seems to bind to the same site on M 3 receptors as KT5720, staurosporine, and WIN 51,708. Therefore, in addition to the allosteric site that binds strychnine (and probably chloromethyl brucine, another allosteric enhancer) there is a second, nonoverlapping, pharmacologically distinct allosteric site on M 3 receptors that also supports positive cooperativity with ACh.

Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of L-[ 3 3 H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M 4 receptors, but not from hippocampal slices, which contain presynaptic M 2 receptors. We conclude that thiochrome is a selective M 4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M 1 to M 3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity.Most receptor-active ligands bind to the same site as the endogenous ligand, the so-called orthosteric site. Agonists mimic the actions of the endogenous ligand, whereas antagonists physically prevent the endogenous ligand from binding but lack its actions. The property of a ligand that determines its effect when bound to a receptor is called its efficacy. Different degrees of efficacy lead to so-called full agonists, partial agonists with a smaller maximal effect than full agonists; neutral antagonists, which occupy the active site without exerting any effect; and inverse agonists, which reduce the activity of constitutively active receptors (Kenakin, 2002). The selectivity of an orthosteric ligand for one receptor or receptor subtype is determined by its affinity for the receptor and its efficacy at that receptor. The difference in affinity between the target receptor and other receptors must be large for an orthosteric ligand to have useful selectivity. This can be difficult to achieve for receptors that show close homology at the orthosteric binding region such as the five subtypes of muscarinic receptor (M 1 -M 5 ) (Hulme et al., 1990). Selectivity derived from efficacy can also be hard to achieve, because the effect of a partial agonist depends on properties of the tissue, such as receptor density and downstream amplification mechanisms, which vary between cells and tissues, so a ligand with no apparent functional effect on tissues in vitro may nevertheless activate tissues in vivo, leading to unacceptable side effects (Terry et al., 2002).An alternative approach for developing selective ligands is to look for allosteric ligands that bind to a site on the receptor which is different from the site to which the endogenous ligand binds. This allows both types of ligand to bind simultaneously. If the affinity (or efficacy) of the endogenous ligand is different when it is bound to the allosteric liganded receptor compared with when it is bound to the free receptor, then the allosteric ligand exhibits positive or negative coop-

We have studied the interactions of five indolocarbazoles with N-[methyl-(3)H]scopolamine (NMS) and unlabeled acetylcholine at M(1)-M(4) muscarinic receptors, using equilibrium and nonequilibrium radioligand binding studies. The results are consistent with an allosteric model in which the primary and allosteric ligands bind simultaneously to the receptor and modify each other's affinities. The compounds were generally most active at M(1) receptors. [(3)H]NMS binding was enhanced by staurosporine, KT5720, and KT5823 at M(1) and M(2) receptors, and by K-252a at M(1) receptors. Gö 7874 reduced [(3)H]NMS affinity by up to threefold for all subtypes. A range of cooperative effects with acetylcholine was seen, and, at the M(1) receptor, KT5720 had a log affinity of 6.4 and enhanced acetylcholine affinity by 40%. The compounds inhibited the dissociation of [(3)H]NMS to different extents across the receptor subtypes, with the largest effects at M(1) receptors. In equilibrium binding studies the inhibitory potency of gallamine at M(1) receptors was not affected by KT5720, indicating that these agents bind to two distinct allosteric sites and have neutral cooperativity with each other. In contrast, gallamine and staurosporine had a negatively cooperative or competitive interaction at M(1) receptors. Similarly, the potency and relative effectiveness of KT5720 for inhibiting [(3)H]NMS dissociation from M(1) receptors were not affected by gallamine or brucine, but were affected in a complex manner by staurosporine. These results demonstrate that there are at least two distinct allosteric sites on the M(1) receptor, both of which can support positive cooperativity with acetylcholine.

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