The size and/or configuration of the cycloalkane D ' ring in pentacyclic progesterone derivatives are crucial for their high-affinity binding to a protein in addition to progesterone receptor in rat uterine cytosol

[H-3]labeled progesterone and a number of its 16 alpha,17 alpha-cycloalkano derivatives with an additional three to six-membered D' ring were investig ated for mutual competition and equilibrium binding to proteins from rat ut erine cytosol. The interaction of all studied [H-3]ligands with proteins wa s characterized by comparable affinity (K-d in nM region) and apparent homo geneity in terms of affinity. At the same time, the concentrations of bindi ng sites for ligands bearing 16 alpha,17 alpha cyclopentano, cyclohexano, o r cyclohexeno substituents were several-fold hi her than those for progeste rone or 16 alpha,17 alpha-cyclopropanoprogesterone. In mutual competition e xperiments, when [H-3]progesterone or [H-3]16 alpha,17 alpha-cyclopropanopr ogesterone were used. the curves of 'bound radioactivity-log of competitor concentration' for ail compounds studied were parallel and corresponded to a model of 'one protein-two ligands.' However, when [H-3]ligands with bulky 16 alpha,17 alpha-substituents (with the possible exception of cyclohexene derivative) were used, competitive curves for various ligands had differen t appearances and fell into two groups. Parallel curves for derivatives wit h 5 or 6 carbons in D' ring described by a model of 'one protein-two ligand s' formed the ist group. The 2nd group comprised curves for progesterone or 16 alpha,17 alpha-cyclopropanoprogesterone that had lower slopes and could be described by a model of 'two proteins-two ligands.' Taken together, the results suggest the presence in rat uterine cytosol, of a protein in addit ion to progesterone receptor capable of discriminating between ligands with no or small 16 alpha,17 alpha-cycloalkano substituents and ligands with mo re bulky substituents. (C) 2000 Elsevier Science Inc. All rights reserved.