E. V. Pokrovskaya

[Structural features of pregna-D'-pentaranes determining their interaction with three rat proteins].

Competition of a number of progesterone 16α,17α-cycloalkane derivatives with 3H-labeled ligands for the binding sites of rat uterine progesterone receptor, uterine pentaranophilin, and blood serum pentaranophilin was studied. We found that the selective ligands for the progesterone receptor are progesterone, 16α,17α-cyclopropanoprogesterone, and 16α,17α-cyclopent-3′-enoprogesterone and the selective ligands for serum pentaranophilin are 6α-methyl-16α,17α-cyclohexanopregna-1,4-diene-3,20-dione and 3β-hydroxy-16α,17α-cyclohexanopregn-5-en-20-one. No selective ligands for the uterine pentaranophilin were found. The majority of substituents in rings A, B, and D′ we studied decreased the affinity of ligands for all the three proteins. The substitution of the Δ5-3β-hydroxy grouping for the Δ4-3-keto grouping exerted the strongest negative effect in the case of the progesterone receptor and the uterine pentaranophilin, whereas the introduction of the 3′,4′-dimethyl grouping strongly inhibited the ligand affinity for the uterine pentaranophilin. The extent and even the direction of the effect of a substituent on the affinity of ligands for the proteins substantially depended on the presence of other substituents in the steroid molecules. We hypothesized that a certain similarity exists between three proteins studied in respect to the structures of their ligand-binding pockets.

Species and Tissue Distribution of Proteins Binding 16α,17α-Cycloalkanoprogesterone Derivatives

The binding of [3H]progesterone and [3H] 16 alpha,17 alpha-cycloalkanoprogesterones to proteins from rat, rabbit, and human uteri and other organs was studied. We found that 16 alpha,17 alpha-cycloalkanoprogesterone derivatives display affinities for the uterine progesterone receptors comparable with that of the natural hormone and no substantial species differences in the affinity. Rabbit uterus was found to have no proteins distinct from the progesterone receptor that specifically bind [3H] 16 alpha,17 alpha-cycloalkanoprogesterones. At the same time, in the human uterus, we found another protein that binds some of these progesterone derivatives; it turned out to be similar to the protein from rat uterus. A similar protein with the same selectivity and affinity for steroids was also found in rat and human kidneys. Blood serum, liver, lung, and a number of other tissues were found to contain a protein of the third type that binds the same 16 alpha,17 alpha-cycloalkanoprogesterones and exhibits submicromolar Kd values for these steroids and a very low affinity for progesterone. We speculated that the introduction of a bulky substituent adjacently to the 17 beta-side chain of progesterone could result in a change in the general biodynamics of the derivative including its transport, uptake, and accumulation in tissues, which may determine the selectivity of its effect.The binding of [3H]progesterone and [3H]16α,17α-cycloalkanoprogesterones to proteins from rat, rabbit, and human uteri and other organs was studied. We found that 16α,17α-cycloalkanoprogesterone derivatives display affinities for the uterine progesterone receptors comparable with that of the natural hormone and no substantial species differences in the affinity. Rabbit uterus was found to have no proteins distinct from the progesterone receptor that specifically bind [3H]16α,17α-cycloalkanoprogesterones. At the same time, in the human uterus, we found another protein that binds some of these progesterone derivatives; it turned out to be similar to the protein from rat uterus. A similar protein with the same selectivity and affinity for steroids was also found in rat and human kidneys. Blood serum, liver, lung, and a number of other tissues were found to contain a protein of the third type that binds the same 16α,17α-cycloalkanoprogesterones and exhibits submicromolar Kd values for these steroids and a very low affinity for progesterone. We speculated that the introduction of a bulky substituent adjacently to the 17β-side chain of progesterone could result in a change in the general biodynamics of the derivative including its transport, uptake, and accumulation in tissues, which may determine the selectivity of its effect.

Synthesis of 19-substituted steroids of the 16α,17α-cyclohexanopregnane series and study of their interactions with rat uterine cytosol and blood serum proteins

New 16α,17α-cyclohexanopregnanes (pregna-D′6-pentaranes) containing alkyl or heteroatomic substituents in position 19, namely, 19-methylidene-, 19-methyl-, 19-methoxyimino-, and 19-(3-methoxycarbonylpropoxyimino)-16α,17α-cyclohexanopregn-4-ene-3,20-diones were synthesized, and their interactions with the progesterone receptor and pentarane-binding proteins of rat uterus cytosol and rat blood serum was studied. The same substituent in position 19 of the steroid molecule can affect in opposite direction its affinity to these proteins. These results suggest differences in the structures of the ligand-binding pockets of the three proteins in the region surrounding the C(10) atom of the steroid nucleus.

Synthesis of 6(E)- and 6(Z)-(3-ethoxycarbonylpropyl)oximes of 16α,17α-cyclohexanopregn-4-ene-3,6,20-trione and study of their interaction with proteins of the rat uterine cytosol and blood serum

Abstract6(E)- and 6(Z)-(3-Ethoxycarbonylpropyl)- and -(3-carboxypropyl)oximes of 16α,17α-cyclohexanopregn-4-ene-3,6,20-trione were synthesized. The reactions of these ester ligands with pentarane-binding proteins of the uterine cytosol and blood serum were studied; the latter exhibits a higher affinity. The preferred binding of the oxime (E)-isomer relative to the (Z)-isomer was noted.

Bioactive 5-Reduced 16α,17α-Cyclohexanoprogesterone Derivatives Weakly Interact with Proteins of the Soluble Uterine Fraction

We studied competitive activities of 16α,17α-cyclohexano-5α- and 5β-dihydroprogesterone in replacing 3H-progesterone and 3H-16α,17α-cyclohexano-6α-methylprogesterone from protein complexes. Direct binding of 3H-5-reduced derivatives with proteins of soluble fractions from rat and rabbit uteri was also assayed. Cd values for 5-reduced derivatives were in the micro- or submicromolar range. The data suggest that biological effects of these analogues are not mediated via soluble uterine receptors.

Interaction between 16α,17α-cyclopropane progesterone and progesterone receptors in rat uterus

Binding of3H-16α, 17α-cyclopropanoprogesterone (CPG) and3H-progesterone (PG) to progesterone receptors in soluble fraction of rat uterus is studied. It is shown that CPG and PG specifically bind to the protein with similar affinity and binding capacity. Unlabeled PG competitively inhibits the binding of CPG, and unlabeled CPG competitively inhibits the binding of PF with the same efficiency. Dissociation of CPG- and PG-receptor complexes is characterized by the same dissociation constant.

Morphological Changes in Rat Uterus Tissues under the Action of Progestins and Antiprogestins of the Pregna-D"-pentaran Series

Changes in the uterus morphology of mature female rats were studied on the model of pseudopregnancyafter treatment with the progestin 16α,17α-cyclohexanoprogesterone (PR) and the antirpogestins 5α(H)-16α,17α-cyclohexano-4,5-dihydroprogesterone (APR1) and 5β(H)-16α,17α-cyclohexano-4,5-dihydroprogesterone (APR2). The rats were preliminarily estrogenized with 17β-estradiol at a dose of 1 μg/(animal day) for 4 days and then treated with PR at a dose of 0.2 mg/(animal day) for 14 days. The first group was then left without any treatment, whereas APR1 and APR2 were injected at the dose of 0.2 mg/(animal day) for 4 days to the animals of the second and the third groups, respectively. Light and electron microscopy of the uterus preparations demonstrated that the PR action provoked a complete pseudopregnancy picture characterized by the endometrium functionalization and the myometrium hypertrophy. Subsequent treatment with APR1 and APR2 caused the hypertrophy to cease, which had a more pronounced effect in the case of APR2. At the same time, some indications of the endometrium functionalization remained observable after treatment with APR1 and APR2. The specific binding sites for 3H-labeled APR1 and APR2 were absent from the uterus cytosol for the rats gestagenized with PR.