Dysfunction of muscarinic cholinergic systems of the central nervous system contributes to the deficits associated with many illnesses, including Alzheimer's Disease. However, treatment with directly-acting muscarinic cholinergic drugs has not been proven to be very beneficial to date. This may relate in part to the adverse effects and lack of selectivity of currently available muscarinic drugs. There are theoretical reasons why allosteric drugs can have inherent advantages of selectivity, efficacy, and safety over directly-acting (competitive) agents; the allosterically- acting benzodiazepines are good examples of CNS drugs that are very effective and safe. Although it is known that muscarinic receptors are susceptible to allosteric regulation, studies of substances which allosterically modulate muscarinic receptors have been hampered by several problems: the specificity of allosteric drugs toward subtypes of muscarinic receptors was difficult to determine in tissues where mixed subtypes were present; it was not clear whether different modulators acted at a single, well-defined, allosteric site; and it has not been possible to label the allosteric site directly. The investigators have shown that all muscarinic receptor subtypes (m1-m5) are subject to allosteric regulation, that there are marked differences between subtypes, and that at least some allosteric modulators interact with a common well-defined allosteric site. They have used molecular genetic techniques to localize regions of the receptors that are responsible for the subtypes selectivities of some allosteric modulators. Further, they have demonstrated that one of these allosteric agents will make a good radiolabled ligand with which to label the allosteric site directly.
The aims of this proposal are to use these tools to characterize the allosteric sites in muscarinic receptor subtypes in great detail: (1) they will screen the growing number of allosteric modulators to determine their subtype-specificities and whether they act at common site(s); (2) they will use mutated receptors to identify the precise receptor sequences responsible for the allosteric effects and selectivity; and (3) they will analyze allosteric modulation of functional responses by sophisticated methods that will allow us to test and refine the allosteric model (studies in other systems have shown that allosteric modulators may antagonize or augment responses). The long-range goals of these studies are the discovery and classification of subtype-selective allosteric agents that modulate muscarinic receptor functions in well-defined ways.
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