We thus provide new insights into structure–activity relationships of steroids . Progesterone production was normalized for the amount of protein in each well. variability in the SAR models or the in vitro assay results themselves must be considered .. downloaded from the EPA website. The structures of .. They were. 17~-estradiol, coumestrol, BPA and progesterone whose RBA values were Apr 9, nor-progesterone (19NP) is a potent progestagen which possesses a high affinity for the progesterone receptor (PgR). In contrast.
The human ER belongs to the ligand-activated nuclear receptor superfamily. Some members of this family are receptors for steroid hormones, vitamin D, retinoic acid, and thyroid hormones 34. A substantial body of research shows that these two receptor subtypes are differentially expressed in various tissues, and each contributes to the overall pharmacology of estrogen 5.
These ERs exist in transcriptionally inactive conformations, and binding with ligands induces an active and stable conformation that allows its dimerization homo or hetero 1. Different ligands appear to interact with the receptor, and each ligand receptor complex can result in a unique conformation that can further interact with or recruit cell and tissue-specific receptor-associated coactivators or corepressor proteins.
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 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.
Finally, and again consistent with the profiles presented in hydroxyl is a more important pharmacophore than the C17 hydro- Fig. The ability of the hydroxylated family of steroids rounding the C3 hydroxyl of cholesterol . In fact, hydroxylation to associate with the CRAC peptide as well as preferentially inhibit at the C3 and C19 carbons appears critical for bioactivity, because mitochondrial cholesterol indicates that this pharmacophore pro- steroid 4which possesses a ketone group at C3 and an aldehyde vides pharmacological selectivity for TSPO-mediated inhibition of at C19, is ineffectual.
Moreover, the observations that hydroxyl- steroid biosynthesis. Discussion motif, findings that will have to be tested with further structural understanding of the protein. Currently, the highest resolution In our efforts to understand the biological functions of the structure of a bacterial TSPO homolog does not provide us with 18 kDa mitochondrial TSPO, a high-affinity drug- and cholesterol- sufficient detail to accurately address this question .
In the current studies, we have investigated to find for an endogenous substituted steroid is 4-androsten- the structure—activity relationships of the substituted diol-3,one , the short-lived first intermediate of the A.
Heat map comparing efficacy and potency of inhibition of each steroid under investigation for dbcAMP- and 22R-HC-mediated steroidogenesis. At left, graphical summary of IC50 and percent inhibition values. See text for exact numbers. Though the substituted 50 steroids are not physiologically prominent, other steroids exam- ined in this study are present in vivo.
Hydroxylation of the steroid 40 backbone at position 16 is critical for the synthesis of estriol , as well as the liver metabolism of steroids , which have been 30 observed to be metabolic products of macrophage steroid metabo- lism . These steroids are unlikely to have a negative role in reg- 20 ulating steroidogenesis, however, as the substituted steroids we examined had low inhibitory activity, other than cytotoxicity 10 at high, non-physiological levels.
In contrast, the 4- 7- and substituted steroids significantly inhibited steroidogenesis, 0 appearing at first, to phenocopy the substituted steroids. How- 1 2 5 8 9 11 ever, upon further examination, it became clear that the principal Fig. Synthetic site of action for these compounds was after the mitochondrial CRAC peptides were incubated with [3H]azidocholesterol either alone, with the transport of cholesterol, as they equivalently inhibited dbcAMP-in- substituted steroids 12 [members of the C19 hydroxyl family], 5 [a member of duced and 22R-HC-supported steroid production, and none exhib- the C11 oxo-family], 8 and 9 [members of the C7 hydroxy family], 11 [member of the C4 hydroxy familyor with saturating concentrations of cholesterol to assess ited association with the recombinant TSPO CRAC motif.
It has non-specific binding. After equilibration, samples were exposed to UV light to been known for some time that the planarity of the sterol back- crosslink [3H]azidocholesterol to the peptide.Estrogen - Reproductive system physiology - NCLEX-RN - Khan Academy
The samples were subsequently bone is critical for entry of steroidal compounds into the CYP11A1 filtered through Whatmann paper and radioactivity counted. The results are binding pocket [30,31], and limited space around carbons 4—6 sug- presented as percent bound [3H]azidocholesterol displaced, with greater displace- ment indicative of greater association with the CRAC motif.
Further studies are needed to examine this hypothesis, but the work here strongly suggests that these steroids aromatization reaction performed by cytochrome P CYP19 do not function through TSPO.
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This final point is of note for the [25,26] aromatase. As the substituted steroids would most study of evolution of sterol recognition, as Lathe in proposed A. Novel androstenetriol interacts with mitochondrial translocator protein and controls steroidogenesis.
J Biol This hypothesis postulated that sterols, enzymatically and non- Chem ; While remodeling and apoptosis in osteoblasts. J Steroid Biochem Mol Biol ; 3—5: The anti-tumor effects of androstene [35,36], and its deep phylogenetic conservation makes it a prime steroids exhibit a strict structure-activity relationship dependent upon the candidate for the understanding of sterol recognition evolution orientation of the hydroxyl group on carbon Chem Biol Drug Des ;74 6: Nucleic Acids Res ;39 Web Server it remains an active area of research.
Peripheral-type In conclusion, although we did not find additional TSPO CRAC benzodiazepine receptor-mediated action of steroidogenic acute regulatory domain ligands with our structure—activity analysis outside of protein on cholesterol entry into Leydig cell mitochondria.
Mol Endocrinol the substituted steroid family, we did demonstrate that a broad ;19 2: Rapid colorimetric assay for cellular growth and survival: J Immunol Methods derivatives hydroxylated at a number of positions can potently ;65 1—2: We expect our findings to provide  Bradford MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal useful insights for drug development for diseases due to excessive Biochem ; Characterization of several clonal lines of cultured Leydig tumor cells: Endocrinology Acknowledgments ; 1: The neuro-steroid, 3beta androstene 17alpha diol exhibits potent cytotoxic effects on human malignant glioma and lymphoma We kindly thank Dr.
This work was supported by ;97 5: Int J Biochem Cell Biol ;42 The Research Institute steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis. Three-dimensional structure of TspO by electron cryomicroscopy of helical crystals. Structural basis for androgen specificity and oestrogen synthesis in human aromatase.
Nature References ; J Steroid human steroidogenesis and its disorders.