title: Allosteric interactions of staurosporine and other indolocarbazoles with N-[methyl-(3)H]scopolamine and acetylcholine at muscarinic receptor subtypes: identification of a second allosteric site.
abstract: 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.
authors.affiliation: MRC Technology, Mill Hill, London, United Kingdom.
authors.forename: S
authors.initials: S
authors.lastname: Lazareno
authors.forename: A
authors.initials: A
authors.lastname: Popham
authors.forename: N J
authors.initials: NJ
authors.lastname: Birdsall
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site; D000818:Animals; D016466:CHO Cells; D002227:Carbazoles / Q000494:pharmacology; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D026121:Indole Alkaloids; D007211:Indoles / Q000494:pharmacology; D019832:N-Methylscopolamine / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000145:classification / Q000187:drug effects / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology*
chemical_list: D002227:Carbazoles; D004791:Enzyme Inhibitors; D026121:Indole Alkaloids; D007211:Indoles; D011758:Pyrroles; D011976:Receptors, Muscarinic; C057416:KT 5720; C049985:staurosporine aglycone; D019311:Staurosporine; D000109:Acetylcholine; D019832:N-Methylscopolamine