title: Use of the radioligand [(125)I]-SB-217644 in the characterisation and solubilisation of the novel binding site for the anticonvulsant SB-204269 in rat brain membranes and cell lines.
abstract: SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3, 4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3R-ol) shows anticonvulsant activity in a range of animal seizure models, with a high therapeutic index and a lack of side-effects. We have previously reported the characterisation of a novel binding site for [(3)H]-SB-204269 in rat forebrain, which has a unique profile unrelated to other known anticonvulsant sites of action. We now describe the use of a [(125)I]-labelled form of SB-217644 (trans-6-acetyl-4S-(3-iodobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol), an analogue of SB-204269, for studies on this novel binding site. In rat forebrain membranes, [(125)I]-SB-217644 shows a similar binding profile to that of [(3)H]-SB-204269, with a maximum specific binding capacity (B(max)) of 286+/-12 fmol/mg protein, but has twenty-fold higher affinity (K(d) value 1.7+/-0.1 nM). The high affinity and high specific activity of [(125)I]-SB-217644 allowed it to be used for detection and characterisation of the detergent-solubilised form of the binding site. Specific [(125)I]-SB-217644 binding to cholate-solubilised rat cerebellum showed a K(d) value of 2.7+/-0.3 nM and a B(max) value of 55+/-11 fmol/mg protein, with a 7.3+/-0.3% yield of solubilised binding sites. [(125)I]-SB-217644 was also used in whole-cell binding assays for investigation of the properties of the novel binding site in a range of cell lines. Both rat brain neuronal and glial primary cultures and several CNS-related cell lines were found to have levels of specific [(125)I]-SB-217644 binding similar to those present in rat forebrain membranes. The solubilisation of this novel binding site, and the ability to quantify and characterise it in solubilised tissues and whole cells using [(125)I]-SB217644, will allow further studies towards the ultimate identification of the molecular target of SB-204269.
authors.affiliation: Department of Neuroscience Research, SmithKline Beecham Pharmaceuticals, Harlow, CM19 5AW, Essex, UK. hugh_herdon-1@sbphrd.com
authors.forename: H J
authors.initials: HJ
authors.lastname: Herdon
authors.forename: F M
authors.initials: FM
authors.lastname: Godfrey
authors.forename: W N
authors.initials: WN
authors.lastname: Chan
mesh_terms: D000818:Animals; D001549:Benzamides / Q000378:metabolism*; D001578:Benzopyrans / Q000378:metabolism* / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D003902:Detergents; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D001549:Benzamides; D001578:Benzopyrans; D003902:Detergents; D007457:Iodine Radioisotopes; C102319:SB 204269; C415429:SB 217644