Structure–activity relationships (SAR) of the contraceptive progestogens for (I) oral contraceptive activity (OCA), ing but readily reversible and virtually free of side effects . molecules included in this study are: (1) progesterone (P), (2). This Master's Thesis is brought to you for free and open access by ThinkIR: variability in the SAR models or the in vitro assay results themselves must be 17~-estradiol, coumestrol, BPA and progesterone whose RBA values were Request PDF on ResearchGate | Quantitative Structure-Activity Relationship Studies of Progesterone Receptor Binding Steroids | The selection of appropriate .
Over 30 different receptor-associated protein cofactors have been identified 1. Inherent in this mechanism is the earlier concept 6 that binding affinity of a ligand for its cognate receptor was the sole determinant of biological activity of the ligand is no longer tenable 17 — 9. Moreover, a large body of evidence now clearly indicates that the steroid hormone receptor pharmacology involves at least three related mechanisms for hormone selectivity 7.
Conjugated equine estrogen CEE preparations are widely used for estrogen replacement therapy and contain sulfate esters of the classical estrogens: The interaction of these estrogens with crude ER preparations that mostly likely contained mixtures of ER subtypes has been previously reported Because each of the aforementioned 11 estrogens is structurally different Fig.
Structures of equine estrogens used in our study. These estrogens, in their sulfate-conjugated forms, are components present in the drug CEE. The unlabeled equine estrogens were authentic samples generously donated by Dr.
Their purity has been verified by infrared, mass, and nuclear magnetic resonance spectroscopy. A number of these have been used in several clinical studies from our laboratory 10 — All other biochemicals and reagents were obtained from various commercial sources. All equine estrogens were purified by recrystallizations, and their identity and purity further confirmed by infrared spectroscopy, melting points, and HPLC.
All aqueous counts were done in Ready Safe liquid scintillation cocktail Beckmanand the nonaqueous counts were determined using toluene phosphor. The dissociation constant Kd was calculated from Scatchard plots as described previously 1415and these experiments were done in quadruplicates. Incubations were performed at 4 C for 18 h, and the bound and free steroids were separated using the hydroxylapatite method Each experiment was repeated two to three times.
The cells were routinely maintained as monolayers in T75 flasks at 37 C.
Transfections were performed in phenol-red free MEM supplemented with 0. For cotransfection experiments, 0.
Cotransfections were also performed with different ratios 1: TSPOa high-affinity drug- and cholesterol-binding protein.
We found that in addition to steroids hydroxylated at carbon C19, hydroxylations at C4, C7, and C11 contributed to inhibition of cAMP-medi- ated steroidogenesis in a minimal steroidogenic cell model.
We thus provide new insights into structure—activity relationships of steroids inhibiting mito- chondrial cholesterol transport and steroidogenic cholesterol metabolic enzymes.
Introduction monophosphate cAMPin response to stimulation by circulating peptide hormones, promotes the traffic of cholesterol from cellular Steroid hormones regulate a wide variety of physiological and stores to the inner mitochondrial membrane .
There, cholesterol developmental processes in vertebrates. Pregnenolone undergoes further metabolic nanthrene ring structure of cholesterol, the metabolic precursor processing by mitochondrial and endoplasmic reticulum cyto- of all steroids .
In these tissues, synthesis of cyclic adenosine chrome P CYP and hydroxysteroid dehydrogenase HSD enzymes to generate the repertoire of steroid hormones . A number of proteins have been found to be critical for diphenyltetrazolium bromide; PBR, peripheral benzodiazepine receptor; TSPO, translocator protein 18kDa. Cell culture membrane space and its C-terminus facing the cytoplasm . Cells were cultured in 75 cm2 cell terol binding to TSPO and plays a role in steroidogenesis [9—11].
This work iden- . In this study, we demonstrate 1 mM dbcAMP or 20 lM 22R-HC in medium in the presence or that steroidogenic inhibitory activity of steroids structurally simi- absence of the steroids under investigation.
All media conditions lar to Atriol varied with the position of hydroxylation of the were serum-free. The dbcAMP stocks were prepared in water. Controls contained equivalent amounts of etha- exhibited affinity for TSPO. Thus, we provide new insights into nol.
At the end of the incubation, culture medium was collected the structure—activity relationships of androstenetriols as modula- and tested for progesterone production using radioimmunoassay tors of steroidogenesis. Progesterone production was normalized for the amount of protein in each well 2. For presentation in figures, the percent change in progesterone production in the pres- The molecule [1,2,6,7-N-3H]progesterone was obtained from ence of drug relative to control values is shown.
The modified cholesterol, 7-azi-5a- lyzed using Prism 4. Cell culture supplies were purchased from Life Technologies Carlsbad, Cell viability and mitochondrial integrity at the end of the incu- CA. Tissue culture plasticware was purchased from Corning Corn- bation protocols described above were assessed using the 3- 4,5- ing, NY.
All compounds used in this study were purchased from dimethylthiazolyl -2,5-diphenyltetrazolium bromide MTT Steraloids, Inc. Newport, RIwith the exception of 5-androsten- Cell Proliferation Kit for mitochondrial integrity  Roche, 3b,17,triol 1whose synthesis is described below. All other Mannheim, Germany. Formazan blue formation was quantified chemicals used were of analytical grade and were obtained from at nm and nm using the Victor quantitative detection various commercial sources. Protein measurement Compound 1 was prepared by a chemical reduction of its Proteins were quantified using the dye-binding assay of Brad- corresponding ketone, 5-androsten-3b,diolone MW ford  with bovine serum albumin BSA as the standard.
Louis, MOwhich reduces aldehydes or ketones to the corre- 2.
The compound 2 3. After all the NaBH4 had been added, the reaction lesterol at a final concentration of nM in the absence or pres- was heated to a boil for 2 min, followed by rapid cooling on ice. After a one lowed to dry. Purity of 1 was determined by mass spectrometry.
Bound radioactivity was deter- used in this study was performed using the ChemMine tools ser- mined by liquid scintillation counting and specific [3H]azidocho- vice . The compound 5-andr- using the Prism 4. Results steroidogenesis than 1 with an IC50 of Though alteration of the 17 position hydroxyl group did Atriol as a TSPO CRAC domain ligand and potent inhibitor of not seem to have a significant effect on steroidogenic inhibition, hormone-stimulated steroidogenesis .
To better understand replacing the 3 position hydroxyl group of 4-androstenb, the structural characteristics of the Atriol molecule, we studied diolone 3 reduced the effectiveness of inhibition, showing no the effect of a spectrum of substituted steroids on steroidogenesis inhibition at 1 lM and reduced inhibition at 10 lM concentrations in the MA Leydig tumor cell model, a minimal steroidogenic cell with an IC50 of Although the 3 position model useful for the study of mitochondrial cholesterol transport hydroxyl of 1 appears important for its inhibitory activity, the .
These steroids were principally differentially modified at hydroxyl group at carbon 19 is absolutely critical, as replacement the [ 1 — 4 ], [ 5 — 7 ], 7-[ 8 — 10 ], 4-[ 11 ,] and [ 12 — of this hydroxyl with an aldehyde group in 4 ablates all inhibitory 15 ] positions of the steroid backbone Fig 1B. Hierarchical clus- activity Fig 2A. These findings indicate that the regions encom- tering of the chemical structures confirmed partitioning of these passing the three hydroxyl group and the 19 hydroxyl group of compounds into four broad categories Fig.
Structures of examined steroids. A Cycloperhydropentanophenanthrene ring structure of steroids with numbered carbons; B examined steroids, 1 5-androsten- 3b,17,triol; 2 5-androsten-3b,diolone; 3 3-androsten,diolone; 4 4-androsten-3,dionealdehyde; 5 5-androsten-3b,11b,17b-triol; 6 4- androsten-3b,17b-diolone; 7 5-androsten-3b,17b-diolone; 8 5-androsten-3b,7a,17b-triol; 9 5-androsten-3b,7b,17b,-triol; 10 5-androsten-3b,7b-diolone; 11 5-androsten-3b,4a,17b-triol; 12 5-androsten-3b,16a,17a-triol; 13 5-androsten-3b,16b,17a-triol; 14 5-androsten-3b,16a,17b-triol; 15 5-androsten-3b,16b,17b- triol; and C computational hierarchical structural clustering of steroids under examination.
Oxygenation at the 4, 7, 11, and 19 carbons of the steroid ring structure acutely inhibits dbcAMP-mediated steroid synthesis. A MA Ledyig tumor cells were incubated for 2 h in the presence of 1 mM dbcAMP and increasing concentrations of substituted steroid.
At the end of incubation, media was collected and progesterone production measured by RIA. Compounds in the figure are indicated by bracketed numbers. B Incubations as in Abut with substituted steroids.
C Incubations as in Abut with 7-substituted steroids. D Incubations as in Abut with 4- and substituted steroids. The ringed sterol backbone of 1 and other steroids are rigidly dbcAMP in the presence of 0— lM of 5-androsten-3b,4a,17b - planar, but the hydroxyl group at carbon 19 would be expected to triol 115-androsten-3b,16a,17a-triol 125-androsten- possess significant conformational flexibility.
To examine the ste- 3b,16b,17a-triol 135-androsten-3b,16a,17b -triol 14and reochemical activity of this region of the molecule, we examined 5-androsten-3b,16b,17b-triol 15 and assessed their steroidogenic the inhibitory effects of 0— lM 5-androsten-3b,11b,17b-triol capacity.
As seen in Fig. Consistent with the small impact diolone 7 on dbcAMP-stimulated progesterone production of substitutions observed with steroids 2 and 10steroids in MA cells. As shown in Fig. These results that the 19 hydroxyl group of 1 is positioned in the region occu- further underscore the importance of the 3-position, and non- pied by the 11 oxygen groups of 5 — 7.
Extended exposure to potent inhibitors of steroidogenesis, with IC50 values of 7-substituted androstenetriols has been demonstrated to be cyto- 2.
Interestingly, steroid toxic to a number of transformed cells [14,20,21]. Though we have 8while differing from 9 and 10 only in the stereochemical previously shown that 1 is not cytotoxic in the time period of orientation of the hydroxyl group at carbon C7, was significantly study, the possibility still existed that the observed inhibitory less effective at inhibiting steroidogenesis, with an IC50 of effects of the remaining compounds could be at least partially Again, these results support the critical nature of related to cytotoxicity.
To this end, MA cells were exposed to a key hydroxyl group extending from the 7 or 11 face of the steroid 0— lM of substituted steroids for 2 h and cellular viability backbone in inhibiting steroidogenesis. Slight inhibition of cellular file Fig. To examine the impact of the 3 and 16 ends of the ste- viability was observed with the ketone derivatives of the roid molecule on steroidogenesis, we incubated MA cells with 7- and substituted steroids Fig.
These results indicate that the moderate activity ob- steroids 5 — 7 significantly inhibited 22R-HC-mediated proges- served for these compounds was due to toxicity for the time period terone production at low micromolar concentrations, with IC50 val- tested. Similar results were obtained with the 7-substituted ste- cAMP-dependent transport of cholesterol into the mitochondria roids 9 and 10 and the 4-substituted 11 Fig.
Summariz- and its subsequent metabolism to progesterone by the mitochon- ing the steroidogenic inhibitory potency and efficacy values of drial CYP11A1 and hydroxysteroid dehydrogenase HSD3b1 . To further our understanding of the ing active substituted steroids, which possessed similar potency effects of these substituted steroids on downstream steroidogenic and efficacy inhibiting dbcAMP- and 22R-HC-supported progester- machinery, we examined the inhibitory effects of 0— lM of one production.
These results suggest that the hydroxylation of the the substituted steroids 1 — 4 on the steroidogenic metabo- steroid ring at carbon C19 provides specificity for inhibition of lism of 22R-hydroxycholesterol 22R-HCan aqueous-soluble cho- mitochondrial cholesterol transport, whereas hydroxylation lesterol analog that is directly metabolized by the mitochondrial of the steroid ring at the 4, 7, and 11 positions confers inhibition CYP11A1  in MA cells.
As previously reported , 5-andros- of cholesterol metabolism. Similar inhibitory profiles were observed cholesterol recognition amino acid CRAC motif at the C-terminus, for compounds 2 and 3 IC50 values: To assess the Effect of oxygenation at the 4, 7, 11, and 19 carbons of the steroid ring structure on acute cellular viability. A MA Ledyig tumor cells were incubated for 2 h in the presence of increasing concentrations of substituted steroid. At the end of incubation, cellular metabolic activity was assessed by MTT assay.
Oxygenation at the 4, 7, 11, and 19 carbons of the steroid ring structure acutely inhibits 22R-HC metabolism. A MA Ledyig tumor cells were incubated for 2 h in the presence of 20 lM 22R-HC and increasing concentrations of substituted steroid. For this assay, recombinant CRAC peptides were 3b,17,triol, 1. The samples were subsequently filtered through What- into mitochondria and metabolism by CYP11A1 to pregeneolone.
The results in Fig. Moreover, supporting the similar pharmacolog- ical properties similar to 1. Of note, 3 was a significantly weaker ical profile, compound 2 was also observed to displace choles- inhibitor of steroidogenesis than 1 or 2indicating that the C3 terol.