title: Analogs of WIN 62,577 define a second allosteric site on muscarinic receptors.
abstract: WIN 51,708 (17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) and WIN 62,577 (17-beta-hydroxy- 17-alpha-ethynyl-delta(4)-androstano[3,2-b]pyrimido[1,2-a]benzimidazole) are potent and centrally active antagonists at rat, but not human, NK(1) receptors. The interactions of these compounds and some analogs with [(3)H]N-methyl scopolamine ([(3)H]NMS) and unlabeled acetylcholine (ACh) at M(1)-M(4) muscarinic receptors have been studied using equilibrium and nonequilibrium radioligand binding methods. The results are consistent with the predictions of the allosteric ternary complex model. The WIN compounds have log affinities for the unliganded receptor in the range 5 to 6.7, and exhibit positive, negative, or neutral cooperativity with [(3)H]NMS and ACh, depending on the receptor subtype and nature of the interacting ligands. WIN 62,577 is an allosteric enhancer of ACh affinity at M(3) receptors. Although interacting allosterically, WIN 62,577 and WIN 51,708 do not affect [(3)H]NMS dissociation from M(3) receptors. Certain analogs have higher affinities than WIN 62,577, and truncated forms of WIN 62,577, including steroids, also act allosterically. One analog, 17-beta-hydroxy-17-alpha-Delta(4)-androstano[3,2-b]pyrido[2,3-b]indole (PG987), has the unique effect of speeding [(3)H]NMS dissociation; its largest effect, 2.5-fold, is at M(3) receptors. The interaction between PG987 and other allosteric agents on [(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.
authors.affiliation: Medical Research Council Technology, Mill Hill, London, UK. slazare@nimr.mrc.ac.uk
authors.forename: S
authors.initials: S
authors.lastname: Lazareno
authors.forename: A
authors.initials: A
authors.lastname: Popham
authors.forename: N J M
authors.initials: NJ
authors.lastname: Birdsall
mesh_terms: D000494:Allosteric Regulation; D000731:Androstanes / Q000494:pharmacology; D000736:Androstenes / Q000494:pharmacology*; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D016466:CHO Cells; D006224:Cricetinae; D008024:Ligands; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000731:Androstanes; D000736:Androstenes; D001562:Benzimidazoles; D008024:Ligands; D011976:Receptors, Muscarinic; C086111:WIN 51708; C086110:WIN 62577