The aim of this proposal is the discovery and classification of subtype selective allosteric agents that modulate muscarinic receptor functions in well-defined ways, with minimal competitive complications. Malfunctions of muscarinic cholinergic systems of the CNS are known or thought to be involved in many diseases, yet treatment with directly acting muscarinic agonists and antagonists has not proven generally beneficial to date. This may relate in part to the adverse effects of currently available muscarinic drugs. Indirectly acting (allosteric) drugs have some inherent advantages of efficacy and safety over directly acting (competitive) agents; the allosterically-acting benzodiazepines are good examples of CNS drugs that are very effective and safe. Studies of substances which allosterically modulate muscarinic receptors have been hampered by three problems: the drugs that have been investigated so far exert some competitive effects as well as allosteric effects; the specificity of these agents toward subtypes of muscarinic receptors has been difficult to determine in tissues where mixed subtypes are present; and, it has been possible to determine whether different modulators act at a single, well defined, allosteric site. Using assays that are not affected by concomitant competitive effects, it has been found that all cloned muscarinic receptor subtypes (m1-m5) are subject to allosteric regulation and that there are marked differences between subtypes. We has also been shown that two allosteric modulators interact in a way that strongly suggests that they bind to the same well-defined allosteric site on cardiac (m2) muscarinic receptors. The proposed studies aim to expand upon these findings. (1) We will screen a growing number of allosteric modulators to determine their subtype-specificities and whether they act at common site(s). (2) We will examine allosteric modulation of functional responses by novel paradigms that are not confound by concomitant competitive interactions. Studies of other allosterically-acting drugs have shown that allosteric modulators may antoagonize or augment responses, or may affect the rate of receptor desensitization. (3) Finally, we will construct chimeric receptors to identify the receptor sequences responsible for the allosteric effects.
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