Ketononestrol aziridine, an agonistic estrogen receptor affinity label: study of its bioactivity and estrogen receptor covalent labeling.
Ketononestrol aziridine [(6R,TS)1-(N-aziridinyl)6,7-bis-(4-hydroxyphenyl)5-nonamone (KNA)], an aziridine derivative of hexestrol, is an estrogenic affinity label for the estrogen receptor (ER). It has an apparent relative binding affinity 8% that of estradiol and shows time-dependent irreversible binding to the ER in uterine cytosol preparations and intact human breast cancer cells (MCF-7). The agonistic activity of KNA is evident in MCF-7 cells in culture, where it increases the cell growth rate and elevates the level of progesterone receptor. KNA was prepared in high specific activity tritium-labeled form by iodination of a methanesulfonate precursor, followed by catalytic tritium-iodine exchange and aziridinylation; the material prepared has high radiochemical purity and a specific activity of 67 Ci/mmol. The covalent attachment of [3H]KNA to the ER can be followed directly by a solvent precipitation assay. In cytosol preparations of uterine ER, labeling with [3H]KNA proceeds in a time-, concentration-, and temperature-dependent manner; labeling is efficient and selective and, by competition studies, was shown to be estrogen specific. ER in intact MCF-7 cells can also be covalently labeled by treatment with [3H]KNA. Receptor covalently labeled with [3H]KNA sediments as a 4S species on high salt sucrose gradients, and its sedimentation position is shifted by treatment with monoclonal antireceptor antibodies. On sodium dodecyl sulfate-polyacrylamide gels, the principal labeled species migrates with a mol wt of 66,000. KNA should prove to be a useful probe for studies on receptor structure, function, and chromatin interactions, particularly when the behavior of a receptor-agonist complex is being investigated.
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001388:Aziridines / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D001389:Azirines / Q000378:metabolism*; D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism; D005260:Female; D006801:Humans; D007659:Ketones / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D011960:Receptors, Estrogen / Q000378:metabolism*; D011980:Receptors, Progesterone / Q000187:drug effects / Q000378:metabolism; D012756:Sheep; D014599:Uterus / Q000378:metabolism
D000345:Affinity Labels; D001388:Aziridines; D001389:Azirines; D007659:Ketones; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; C052977:ketononestrol aziridine; D004958:Estradiol