Structural Features of Pregna-D′-pentaranes Determining Their Interaction with Three Rat Proteins

Competition of a number of progesterone 16,17-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, 16,17-cyclopropanoprogesterone, and 16,17-cyclopent-3-enoprogesterone and the selective ligands for serum pentaranophilin are 6-methyl-16,17-cyclohexanopregna-1,4-diene-3,20-dione and 3-hydroxy-16,17-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 ⁵-3-hydroxy grouping for the ⁴-3-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.     

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

[(3)H]labeled progesterone and a number of its 16alpha, 17alpha-cycloalkano derivatives with an additional three to six-membered D' ring were investigated for mutual competition and equilibrium binding to proteins from rat uterine cytosol. The interaction of all studied [(3)H]ligands with proteins was characterized by comparable affinity (K(d) in nM region) and apparent homogeneity in terms of affinity. At the same time, the concentrations of binding sites for ligands bearing 16alpha,17alpha cyclopentano, cyclohexano, or cyclohexeno substituents were several-fold higher than those for progesterone or 16alpha, 17alpha-cyclopropanoprogesterone. In mutual competition experiments, when [(3)H]progesterone or [(3)H]16alpha, 17alpha-cyclopropanoprogesterone were used, the curves of 'bound radioactivity-log of competitor concentration' for all compounds studied were parallel and corresponded to a model of 'one protein-two ligands.' However, when [(3)H]ligands with bulky 16alpha, 17alpha-substituents (with the possible exception of cyclohexene derivative) were used, competitive curves for various ligands had different appearances and fell into two groups. Parallel curves for derivatives with 5 or 6 carbons in D' ring described by a model of 'one protein-two ligands' formed the 1st group. The 2nd group comprised curves for progesterone or 16alpha, 17alpha-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 addition to progesterone receptor capable of discriminating between ligands with no or small 16alpha, 17alpha-cycloalkano substituents and ligands with more bulky substituents.     

Species and tissue distribution specificity of proteins binding 16alpha,17alpha-cycloalkane derivatives of progesterone

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.     

3- and 19-oximes of 16alpha,17alpha-cyclohexanoprogesterone derivatives: synthesis and interactions with progesterone receptor and other proteins

Series of 3- and 19-oximes of 16alpha,17alpha-cyclohexanoprogesterone derivatives (pregna-d'-pentaranes) have been synthesized with the aim of probing the surfaces of progesterone receptor's and two other protein ligand binding pockets neighboring to 3- and 19-positions of steroid core. The same derivatives were also studied as possible intermediates for attachment to matrixes. The data on affinity constants suggest the presence of hydrophobic cavities with hydrophilic necks in the progesterone receptor and serum pentaranophylin near C19 of bound ligand and the lack of such a cavity in uterine pentaranophylin. Any of 3-oxime substitutions were found to significantly diminish the ligand affinity for the progesterone receptor. It was also found that some of these modifications, in the Z-configuration particularly, might increase the affinity for serum and uterine pentaranophylins. The latter finding suggests the presence of large cavities near C3 of bound ligand in these proteins and interchangeability between 3-keto and 3-oxime groups in ligand-protein interactions.     

Uptake of three [3H]progestins by target tissues in vivo: Implications for the design of diagnostic imaging agents

We have investigated the tissue distribution of radioactivity for 0.5–4 h follwing the i.v. injection of three tritium-labeled progestins in estrogen-primed, immature rats. Whereas [³H]progesterone shows minimal uterine uptake (<0.7% injected dose per gram; %ID/g), the two higher affinity, synthetic progestins [³H]R 5020 (promegestrone) and [³H]ORG 2058 show highly selective uptake that reaches 4–5% ID/g by 1–3 h. The uterus to non-target tissue activity ratio at 2–4 h is approximately 12–20 for R 5020 and ORG 2058, but less than 2 for progesterone; the uterus to blood activity ratio for R 5020 is also high (approximately 15), but is lower for ORG 2058, possibly due to the accumulation of radiolabeled metabolites in the blood. The uterine uptake is selectively blocked by simultaneous injection of a large dose of unlabeled steroid, indicating that the uptake is mediated by a high affinity, low capacity binding system, presumably the progesterone receptor. Pronounced uptake is also observed by the liver and into fat, but is not receptor-mediated. The highly selective target tissue uptake by the two synthetic steroids, but not by progesterone, indicates that one must have ligands with sufficiently high affinity for the target tissue receptor, as well as low affinity for certain non-receptor binding proteins, in order to obtain adequate contrast between target and non-target tissues in dynamic uptake studies. These guidelines will be important in the development of suitable in vivo imaging agents based on the progesterone receptor.     

DNA slows dissociation of progesterone receptor-steroid ligand complexes

Steroid ligands are known to affect the interactions of their respective receptors with DNA. In the present study, the possibility of DNA interference in progesterone receptor-ligand interactions was investigated. An oligonucleotide containing a hormone response element (HRE) was shown to decrease the dissociation rate of complexes of [3H]progesterone or [3H]16alpha,17alpha-cycloalkanoprogesterones with PRs from rabbit and rat uterine cytosol. The extent to which the oligonucleotide affected the dissociation constant varied from about 4- to 1.5-fold depending on the ligand structure and was ranked in the following order: progesterone>16alpha,17alpha-cyclopropanoprogesterone approximately 16alpha,17alpha-cyclopentanoprogesterone>/=16alpha,17alpha-cyclohex-2'-enoprogesterone approximately 6alpha-methyl-16alpha,17alpha-cyclohexanoprogesterone>/=16alpha,17alpha-cyclohexanoprogesterone. The control oligonucleotide lacking HRE had a weak effect, if any, on the dissociation kinetics. No influence of the HRE-containing oligonucleotide on the equilibrium binding of ligands to PR was observed. The results suggest that the DNA partner affects binding of PR to its ligand.     

Approaches to the design of selective ligands for membrane progesterone receptor alpha

A number of progesterone derivatives were assayed in terms of their affinity for recombinant human membrane progesterone receptor alpha (mPRα) in comparison with nuclear progesterone receptor (nPR). The 16α,17α-cycloalkane group diminished an affinity of steroids for mPRα without significant influence on affinity for nPR, thus rendering a prominent selectivity of ligands for nPR. On the contrary, substitution of methyl at C10 for ethyl or methoxy group moderately increased the affinity for mPRα and significantly lowered the affinity for nPR. A similar but even more prominent effect was observed upon substitution of the 3-oxo group for the 3-O-methoxyimino group. A significant preference towards mPRα was also rendered by the 17α-hydroxy group and additional C6–C7-double bond. The data suggest that the modes of lig- and interaction with mPRα and nPR in the C3 region of the steroid molecule are different. One can speculate that combination of the above substitutions at C17, C10, C6, and C3 may give ligand(s) with high specificity towards mPRα over nPR.     

Synthesis, pharmacological evaluation, and structure-activity relationships of benzopyran derivatives with potent SERM activity

The synthesis, binding affinity for estrogen receptor subtypes (ER alpha and ER beta) and pharmacological activity on rat uterus of a new class of potent ligands, characterized by a 3-phenylbenzopyran scaffold with a basic side chain in position 4, are reported. Some of these compounds, endowed with very high receptor affinity, showed potent inhibition of agonist-stimulated uterine growth, with no or limited proliferative effect. Binding affinity mostly depended on the nature and position of substituents at the 3-phenyl ring, while the uterine activity seems to be affected by basic chain length. Compound 9c (CHF4227) showed excellent binding affinity and antagonist activity on the uterus. The docking of benzopyran derivatives explained the structure-affinity relationships observed for 3-phenyl substitution: a small, hydrophobic 4'-substituent could interact with a small accessory binding cavity, while di-substitution at 4' and 3' led to some ER alpha selectivity. This selectivity can be ascribed to differences in amino acid composition and side chain conformation in the region accommodating the 3-phenyl ring at human ER alpha and ER beta ligand-binding domain.     

Interaction of sex steroids with proteins from the soluble fraction of swine and cattle liver (a preliminary analysis)]

The interactions of [3H]estradiol, [3H]testosterone and [3H]progesterone with soluble proteins from porcine and calf liver were studied. The specific binding of [3H]progesterone and [3H]testosterone in calf liver cytosol seems to be due to serum transcortin or its intracellular precursor (analog). Contrariwise, the specific binding of [3H]progesterone observed in porcine liver cytosol was absent in the serum. This binding was characterized by slow association and dissociation dynamics, moderate affinity for the [3H]-ligand and a high binding capacity. The structural determinants of the ligands were studied by competitive inhibition of the [3H]-ligand binding. The delta 4-3-keto group in the steroid A-ring was found to be the most important determinant. An intensive metabolism of [3H]progesterone was observed during its incubation with cytosol (data from thin-layer chromatography). A 3H-metabolite (presumably, 20 beta-dihydroprogesterone) was predominant in the bound ligand fraction. The data obtained suggest that proteins of a steromodulin type are widely distributed in the mammalian liver.     

Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems.

Progesterone and several progesterone metabolites are capable of inhibiting uterine contractility. Some progesterone metabolites have shown little or no affinity for the progesterone receptor but have been found to be potent modulators of the GABAA receptor system. This study examined whether the inhibition of uterine contraction by progesterone and its metabolites was progesterone receptor-mediated or gamma amino butyric acidA (GABAA) receptor-mediated. Uterine contractions were measured in annular rings of uterine tissue, 5 mm in length, from diestrous II rats, under a fixed tension of 1 gram. The steroids tested were 3 beta-hydroxy-5 beta-pregnan-20-one (6 micrograms/ml), 5 beta-pregnane-3,20-dione (10 micrograms/ml), 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha,5 alpha-THP, 27.5 micrograms/ml), and progesterone (40 micrograms/ml). All compounds significantly inhibited spontaneous uterine contractions when compared to controls. No effect was seen by either 16 micrograms/ml of the progesterone antagonist, RU486, or 32 micrograms/ml of the GABAA antagonist, pictrotoxin, when administered alone. However, when uterine tissues were exposed to a combination of the steroid and the antagonist, the effect of 3 beta-hydroxy-5 beta-pregnan-20-one and 3 alpha,5 alpha-THP was blocked by picrotoxin but not by RU486, indicating that the action of these steroids was mediated through the GABAA system. The effect of 5 beta-pregnane-3,20-dione and progesterone was effectively blocked by RU486 but not by picrotoxin, suggesting that their actions were mediated through the progesterone receptor system. These results indicate that multiple mechanisms exist in the uterus for inhibiting uterine contractility by progesterone and its metabolites.     

Inhibition of uterine prostaglandin-F2 alpha production by bovine conceptus secretory proteins

The effect of bovine conceptus secretory proteins (CSP) on uterine prostaglandin (PG)-F2 alpha production was evaluated in dairy cattle following injection of estradiol-17 beta. Intrauterine injections of dialyzed serum proteins (Control, n = 5) or CSP (n = 5) were administered from days 15 through 18 post-estrus. Following intrauterine treatments on day 18, all cows were injected with E2 (3 mg) to stimulate uterine PGF2 alpha production. Plasma concentrations of progesterone (P4) and 15-keto-13,14-dihydro-PGF2 alpha (PGFM) were determined by RIA. The PGFM responses following E2 challenge were decreased (p less than 0.01) for cows receiving CSP versus serum proteins into the uterine lumen. Individual PGFM, P4 and cycle length responses are discussed. Data suggest that proteins secreted by the bovine conceptus suppress uterine PGF2 alpha production during pregnancy recognition in the cow.     

Structural requirements for the action of steroids as quenchers of albumin fluorescence.

1. Androgens, corticoids, gestagens, estrogens and related steroids are effective quenchers of the intrinsic fluorescence of bovine serum albumin. The quenching effect involves the formation of a steroid albumin complex which formation constant (Kf) and free energy of formation (delta G 0) can be determined by fluorescence titration. The fluorimetrically determined delta G 0 values range from -6.5 to -7.5 kcal/mol. 2. 5 alpha-Androstane and 5 alpha-pregnane are effective quenchers of albumin fluorescence, in accord with the essentially hydrophobic nature of the steroid-albumin interaction. Introduction of hydroxy or oxo groups in 5 alpha-androstane decreases the fluorescence quenching action, but the effect of each group declines when other polar groups are present in the steroid molecule. Similar effects occur with 5 alpha-pregnane except that 20-hydroxy (or oxo) duo-polar derivatives are more effective than the parent hydrocarbon. 3. Comparison of delta G 0 values for steroids differing in a single grouping shows that the steroid-albumin interaction is increased by (a) the benzenoid A-ring; (b) sulfate or carboxylate ions in the vicinity of C-3; (c) the 3-oxo group in place of the 3 alpha-hydroxyl (with 5 beta-pregnane derivatives; not with 5 alpha-androstane derivatives); (d) 17 beta-acetyl or 17 beta-hydroxyethyl residues; (e) acetylated or propionated 17 beta-hydroxy groups; (f) acetylated or methylated hydroxy groups at the C-3 of estrogens; (g) delta 5 and delta 6 double bonds; and (h) the 19 beta-methyl group. The maximal variation of delta G 0 determined by affinity-enhancing groups is -0.8 kcal/mol. Conversely, the steroid-albumin interaction is decreased by introduction of (i) oxygen atoms at C-3, C-6, C-11, C-16, and C-17; (j) 17 alpha-ethynyl and 17 alpha-acetoxyl residues; (k) benzoylated or hexahydro-benzoylated beta-hydroxy groups at C-17; (l) acetylated and benzoylated hydroxy groups at C-3; and delta 1 (conjugated) double bond. Oxo groups at C-3, C-6, C-16 and the 16 alpha, 17 alpha-epoxy group are more effective than the corresponding alpha-hydroxyl in decreasing affinity, while at C-11 and C-17, the alpha-hydroxyl is more effective than the beta-hydroxyl and the oxo group. The effect of substituents is influenced by the whole molecular structure, particularly, by the stereostructure at the A/B juncture, and the presence of an oxo group at C-17. 4. The stereospecific effect of substituents at different positions in the steroid molecule suggests that with non-aromatic, A/B trans (planar) steroids, binding to albumin primarily involves the (alpha) rear surface of the B-, C- and D-ring, and possibly, the 17 beta-side chain. With estrogens and A/B cis (dihedral) steroids, the benzenoid A-ring and electron attracting groups at C-3, respectively, may participate in binding.     

Nonsteroidal progesterone receptor ligands (I): synthesis and SAR of new tetrahydronaphthofuranone derivatives

We have synthesized a series of nonsteroidal progesterone receptor (PR) ligands, tetrahydronaphthofuranones, structurally based on the fungal metabolite PF1092C. Structure-activity relationship studies revealed that substituents at the 6- and 7-positions were critical for PR binding affinity and for agonist or antagonist activity. Compounds in this series, exemplified by 19i, exhibited high affinity and high specificity for PR over other steroid hormone receptors and acted as selective PR antagonists. Further modification of PF1092C may generate compounds of potential pharmacological interest.     

Development of a 3D pharmacophore for nonspecific ligand recognition of alpha1, alpha2, alpha3, alpha5, and alpha6 containing GABA(A)/benzodiazepine receptors

Transfected cells containing GABA(A) benzodiazepine receptors (BDZRs) have been utilized to systematically determine the affinity of ligands at alpha1, alpha2, alpha3, alpha5 and alpha6 subtypes in combination with beta2 and gamma2. All but a few of the ligands thus far studied have relatively high affinities for each of these alpha subtype receptors. Thus, these ligands must contain common stereochemical properties favorable for recognition by each of the subtype combinations. In the present work, such a common three-dimensional (3D) pharmacophore for recognition of alpha1, alpha2, alpha3, alpha5 and alpha6 containing GABA(A)/BDZRs types of receptors has been developed and assessed, using as a database receptor affinities measured in transfected cells for 27 diverse compounds. The 3D-recognition pharmacophore developed consists of three proton accepting groups, a hydrophobic group, and the centroid of an aromatic ring found in a common geometric arrangement in the 19 nonselective ligands used. Three tests were made to assess this pharmacophore: (i) Four low affinity compounds were used as negative controls, (ii) Four high affinity compounds, excluded from the pharmacophore development, were used as compounds for pharmacophore validation, (iii) The 3D pharmacophore was used to search 3D databases. The results of each of these types of assessments provided robust validation of the 3D pharmacophore. This 3D pharmacophore can now be used to discover novel nonselective ligands that could be activation selective at different behavioral end points. Additionally, it may serve as a guide in the design of more selective ligands, by determining if candidate ligands proposed for synthesis conform to this pharmacophore and selecting those that do not for further experimental assessment.     

Molecular determinants of non-specific recognition of delta, mu, and kappa opioid receptors

Identification of the molecular determinants of recognition common to all three opioid receptors embedded in a single three-dimensional (3D) non-specific recognition pharmacophore has been carried out. The working hypothesis that underlies the computational study reported here is that ligands that bind with significant affinity to all three cloned opioid receptors, delta, mu, and kappa, but with different combinations of activation and inhibition properties at these receptors, could be promising behaviorally selective analgesics with diminished side effects. The study presented here represents the first step towards the rational design of such therapeutic agents. The common 3D pharmacophore developed for recognition of delta, mu, and kappa opioid receptors was based on the receptor affinities determined for 23 different opioid ligands that display no specificity for any of the receptor subtypes. The pharmacophore centers identified are a protonated amine, two hydrophobic groups, and the centroid of an aromatic group in a geometric arrangement common to all 23, non-specific, opioid ligands studied. Using this three-dimensional pharmacophore as a query for searching 3D structural databases, novel compounds potentially involved in non-specific recognition of delta, mu, and kappa opioid receptors were retrieved. These compounds can be valuable candidates for novel behaviorally selective analgesics with diminished or no side effects, and thus with potential therapeutic usefulness.     

Affinity of 3-acyl substituted 4-quinolones at the benzodiazepine site of GABA(A) receptors

The finding that alkyl 1,4-dihydro-4-oxoquinoline-3-carboxylate and N-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxamide derivatives may be high-affinity ligands at the benzodiazepine binding site of the GABA(A) receptor, prompted a study of 3-acyl-1,4-dihydro-4-oxoquinoline (3-acyl-4-quinolones). In general, the affinity of the 3-acyl derivatives was found to be comparable with the 3-carboxylate and the 3-carboxamide derivatives, and certain substituents (e.g., benzyl) in position 6 were again shown to be important. As it is believed that the benzodiazepine binding site is situated between an alpha- and a gamma-subunit in the GABA(A) receptor, selected compounds were tested on the alpha(1)beta(2)gamma(2s), alpha(2)beta(2)gamma(2s) and alpha(3)beta(2)gamma(2s) GABA(A) receptor subtypes. The 3-acyl-4-quinolones display various degrees of selectivity for alpha(1)- versus alpha(2)- and alpha(3)-containing receptors, and high-affinity ligands essentially selective for alpha(1) over alpha(3) were developed.     

Role of nonhuman primate models in the discovery and clinical development of selective progesterone receptor modulators (SPRMs)

Selective progesterone receptor modulators (SPRMs) represent a new class of progesterone receptor ligands that exert clinically relevant tissue-selective progesterone agonist, antagonist, partial, or mixed agonist/antagonist effects on various progesterone target tissues in an in vivo situation depending on the biological action studied. The SPRM asoprisnil is being studied in women with symptomatic uterine leiomyomata and endometriosis. Asoprisnil shows a high degree of uterine selectivity as compared to effects on ovulation or ovarian hormone secretion in humans. It induces amenorrhea and decreases leiomyoma volume in a dose-dependent manner in the presence of follicular phase estrogen concentrations. It also has endometrial antiproliferative effects. In pregnant animals, the myometrial, i.e. labor-inducing, effects of asoprisnil are blunted or absent. Studies in non-human primates played a key role during the preclinical development of selective progesterone receptor modulators. These studies provided the first evidence of uterus-selective effects of asoprisnil and structurally related compounds, and the rationale for clinical development of asoprisnil.     

(4-Carboxamido)phenylalanine is a surrogate for tyrosine in opioid receptor peptide ligands

(S)-4-(Carboxamido)phenylalanine (Cpa) is examined as a bioisosteric replacement for the terminal tyrosine (Tyr) residue in a variety of known peptide ligands for the mu, delta and kappa opioid receptors. The Cpa-containing peptides, assayed against cloned human opioid receptors, display comparable binding affinity (Ki), and agonist potency (EC50) to the parent ligands at the three receptors. Cpa analogs of delta selective peptides show an increase in delta selectivity relative to the mu receptor. Cpa is the first example of an amino acid that acts as a surrogate for Tyr in opioid peptide ligands, challenging the long-standing belief that a phenolic residue is required for high affinity binding.