Repurposing steroidogenesis inhibitors for the therapy of neuropsychiatric disorders: Promises and caveats.

Steroids exert a profound influence on behavioral reactivity, by modulating the functions of most neurotransmitters and shaping the impact of stress and sex-related variables on neural processes. This background - as well as the observation that most neuroactive steroids (including sex hormones, glucocorticoids and neurosteroids) are synthetized and metabolized by overlapping enzymatic machineries - points to steroidogenic pathways as a powerful source of targets for neuropsychiatric disorders. Inhibitors of steroidogenic enzymes have been developed and approved for a broad range of genitourinary and endocrine dysfunctions, opening to new opportunities to repurpose these drugs for the treatment of mental problems. In line with this idea, preliminary clinical and preclinical results from our group have shown that inhibitors of key steroidogenic enzymes, such as 5α-reductase and 17,20 desmolase-lyase, may have therapeutic efficacy in specific behavioral disorders associated with dopaminergic hyperfunction. While the lack of specificity of these effects raises potential concerns about endocrine adverse events, these initial findings suggest that steroidogenesis modulators with greater brain specificity may hold significant potential for the development of alternative therapies for psychiatric problems. This article is part of the Special Issue entitled 'Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders'.

Onset of puberty and ovarian steroidogenesis following adminstration of methanolic extract of Cuscuta reflexa Roxb. stem and Corchorus olitorius Linn. seed in mice.

The effect of methanolic extract (ME) of Cuscuta reflexa stem Roxb. and Corchorus olitorius Linn. seed on the onset of reproductive maturity and the ovarian steroidogenesis was studied by means of biochemical techniques. ME of Cuscuta reflexa stem and Corchorus olitorius seed treatment causes a remarkable delay in sexual maturation as evidenced by the age at vaginal opening and appearance of first estrus (cornified smear). The same treatment also results in a significant diminution of Delta(5)-3beta-hydroxysteroid dehydrogenase (HSD) and glucose-6-phosphate dehydrogenase (G-6-PD) activity along with a reduction in the weight of ovary, uterus and pituitary. On the basis of above data, it is assumed that the probable cause of delayed maturation in ME of Cuscuta reflexa stem and Corchorus olitorius seed treated mice is due to the suppressed ovarian steroidogenesis.

Anti-steroidogenic activity of methanolic extract of Cuscuta reflexa roxb. stem and Corchorus olitorius Linn. seed in mouse ovary.

Methanolic extract (ME) of both C. reflexa stem and C. olitorius seed arrested the normal oestrus cycle of adult female mouse and significantly decreased the weight of ovaries and uterus. The cholesterol and ascorbic acid contents in ovaries were significantly increased in the treated mice. Two key enzymes, delta5-3beta-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase, were decreased significantly in ME of both C. reflexa stem and C. olitorius seed after 17 days of treatment. High level of substrates and low level of enzymes indicate the inhibition of steroidogenesis in treated mice and may be due to the presence of flavonoids.

Neuropeptide Y inhibits the biosynthesis of sulfated neurosteroids in the hypothalamus through activation of Y(1) receptors.

We have recently shown that hydroxysteroid sulfotransferase (HST), the enzyme responsible for the biosynthesis of pregnenolone sulfate (Delta(5)PS) and dehydroepiandrosterone sulfate (DHEAS), is expressed in neurons located in the anterior preoptic area and the dorsal magnocellular nucleus of the frog diencephalon. As these two nuclei are richly innervated by NPY-immunoreactive fibers, we investigated the possible implication of NPY in the control of Delta(5)PS and DHEAS biosynthesis. Double labeling of frog brain sections revealed that 42% of the HST-immunoreactive perikarya in the diencephalon were contacted by NPY-containing fibers. In situ hybridization studies showed that Y(1) and Y(5) receptor mRNAs are expressed in the anterior preoptic area and the dorsal magnocellular nucleus. Pulse-chase experiments with (35)S-labeled 3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor demonstrated that frog NPY (fNPY) inhibited the conversion of [(3)H]Delta(5)P and [(3)H]dehydroepiandrosterone ([(3)H]DHEA) into [(3)H,(35)S]Delta(5)PS and [(3)H,(35)S]DHEAS by diencephalic explants. The inhibitory effect of fNPY on Delta(5)PS and DHEAS formation was mimicked by (pPYY) and [Leu(31),Pro(34)]pNPY, which is an agonist for non-Y(2) receptors in mammals, and was completely suppressed by the Y(1) receptor antagonist BIBP3226. Conversely, the Y(2) receptor agonist pNPY-(13-36) and the Y(5) receptor agonist [D-Trp(32)]pNPY did not significantly modify the biosynthesis of [(3)H,(35)S]Delta(5)PS and [(3)H,(35)S]DHEAS. The present study provides the first evidence for the innervation of neurosteroid-producing neurons by NPY fibers. Our data also demonstrate that NPY, acting via Y(1) receptors, exerts an inhibitory effect on the biosynthesis of sulfated neurosteroids.

Common phytochemicals are ecdysteroid agonists and antagonists: a possible evolutionary link between vertebrate and invertebrate steroid hormones.

Many plant compounds are able to modulate growth and reproduction of herbivores by directly interacting with steroid hormone systems. In insects, several classes of phytochemicals, including the phytoestrogens, interfere with molting and reproduction. We investigated whether the anti-ecdysone activity may be due to interaction with the ecdysone receptor (EcR) using a reporter-gene assay and a cell differentiation assay of an ecdysone-responsive cell line, Cl.8+. We tested rutin (delays molt in insects); four flavones: luteolin and quercetin (metabolites of rutin), and apigenin and chrysin; and three non-flavones, coumestrol and genistein (both estrogenic) and tomatine (alters molt in insects). None of the phytochemicals tested were ecdysone agonists in the reporter-gene assay, but the flavones were able to significantly inhibit EcR-dependent gene transcription. In the Cl.8+ cells, quercetin and coumestrol were mixed agonists/antagonists, while genistein, tomatine and apigenin showed a synergistic effect with ecdysteroid in the reduction of cell growth. We suggest that the rutin effects on molting in insects are most likely due to the metabolites, luteolin or quercetin, while tomatine acts via a non-EcR pathway. Flavones not only interact with EcR and estrogen receptor (ER), but also signal nitrogen-fixing bacteria to form root nodules. The NodD protein which regulates this symbiosis has two ligand-binding domains similar to human ERalpha. The evolutionary significance of these findings are discussed.

Assessment of natural products in the Drosophila melanogaster B(II) cell bioassay for ecdysteroid agonist and antagonist activities.

Ecdysteroid agonist and antagonist activities can be detected and quantified with the Drosophila melanogaster B(II) cell bioassay. This bioassay is convenient, sensitive and robust. We report the assessment with this bioassay of the activities of a wide range of compounds representing a number of classes of natural products. Many compounds were inactive over a wide concentration range (10(-8) to 10(-4) or 10(-3) M) or cytotoxic at high concentrations. However, antagonisitic activity was associated with several classes of compounds: cucurbitacins and withanolides (extending previous findings) and phenylalkanoids and certain alkaloids (described for the first time). A withanolide (withaperuvin D) is identified which possesses agonistic activity. Brassinosteroids, which have been ascribed (ant)agonistic properties in the past, were not found to be active in the B(II) bioassay, either as agonists or antagonists. Possible reasons for the prevalence of antagonists and for the low potency of the majority of them are discussed.

gamma-Aminobutyric acid, acting through gamma -aminobutyric acid type A receptors, inhibits the biosynthesis of neurosteroids in the frog hypothalamus.

Most of the actions of neurosteroids on the central nervous system are mediated through allosteric modulation of the gamma-aminobutyric acid type A (GABA(A)) receptor, but a direct effect of GABA on the regulation of neurosteroid biosynthesis has never been investigated. In the present report, we have attempted to determine whether 3beta-hydroxysteroid dehydrogenase (3beta-HSD)-containing neurons, which secrete neurosteroids in the frog hypothalamus, also express the GABA(A) receptor, and we have investigated the effect of GABA on neurosteroid biosynthesis by frog hypothalamic explants. Double immunohistochemical labeling revealed that most 3beta-HSD-positive neurons also contain GABA(A) receptor alpha(3) and beta(2)/beta(3) subunit-like immunoreactivities. Pulse-chase experiments showed that GABA inhibited in a dose-dependent manner the conversion of tritiated pregnenolone into radioactive steroids, including 17-hydroxy-pregnenolone, progesterone, 17-hydroxy-progesterone, dehydroepiandrosterone, and dihydrotestosterone. The effect of GABA on neurosteroid biosynthesis was mimicked by the GABA(A) receptor agonist muscimol but was not affected by the GABA(B) receptor agonist baclofen. The selective GABA(A) receptor antagonists bicuculline and SR95531 reversed the inhibitory effect of GABA on neurosteroid formation. The present results indicate that steroid-producing neurons of the frog hypothalamus express the GABA(A) receptor alpha(3) and beta(2)/beta(3) subunits. Our data also demonstrate that GABA, acting on GABA(A) receptors at the hypothalamic level, inhibits the activity of several key steroidogenic enzymes, including 3beta-HSD and cytochrome P450(C17) (17alpha-hydroxylase).

Ex vivo and in vitro testis and ovary explants: utility for identifying steroid biosynthesis inhibitors and comparison to a Tier I screening battery.

Testis and ovary explants have been proposed as in vitro screens for identifying potential inhibitors of steroid biosynthesis. The goals of the current study were to optimize the conditions of the two assays, to characterize these assays using several compounds with well-defined endocrine activity, and to compare the responses from the explant assays with an in vivo male battery currently undergoing validation using the Crl:CD BR rat in order to evaluate their utility as test systems for screening unknown compounds for possible steroid biosynthesis inhibition activity. There were two components to the testis/ovary assays: ex vivo and in vitro. The ex vivo component used testes/ovaries from animals dosed with the test compounds in vivo, and the in vitro component used testes/ovaries from control animals. For the testis assays, decapsulated testis explants (50 mg) were placed into glass scintillation vials, +/-1.0 IU/ml hCG for 3 h in a shaking water bath (34 degrees C). Following the incubation period, medium was removed, centrifuged, and frozen until assayed for hormone concentrations. A similar procedure was used for the ovary explant assay except that each ovary was incubated separately. The testis explants were evaluated using the following compounds: ketoconazole (KETO), a testosterone biosynthesis inhibitor; aminoglutethimide (AG) (only in vitro) and anastrozole (ANA), aromatase inhibitors; finasteride (FIN), a 5alpha-reductase inhibitor; 17beta-estradiol (17beta-E2), an estrogen receptor agonist; flutamide (FLUT), an androgen receptor antagonist; ICI-182,780 (ICI), an estrogen receptor antagonist; haloperidol (HALO), a D2 receptor antagonist; and reserpine (RES), a dopamine depletor. In the ovary assay, AG (only in vitro), ANA, ICI, and HALO (only in vitro) were evaluated. Addition of fetal calf serum to the medium allowed measurement of estradiol (E2) in the testis assay, but production was not inhibited by ANA or AG. In the ovary explant assay, only AG was identified as inhibiting E2 production in vitro. Hence, both the testis and ovary explant assays appear to have limited utility for detecting aromatase inhibitors. Screening of these nine diverse endocrine-active compounds resulted in all of them being identified as altering the endocrine system when assessed by ex vivo and in vitro testis explants. Using only the in vitro assessment with the criteria of steroid biosynthesis inhibition, four of nine compounds were correctly identified in the testis explant assay (17beta-E2, KETO, FLUT, and HALO). The predictability of both the in vitro and ex vivo ovary assay was 50%, suggesting a 50% false positive or negative rate with unknown compounds. However, of the seven compounds assessed to date (17beta-E2, ICI, ANA, KETO, FLUT, HALO, and RES), all were correctly identified using an in vivo male battery, which also has the capability to detect other endocrine activities. Therefore, the testis and ovary explant assay would not be necessary if one were using an in vivo male battery, since this screen would identify steroid biosynthesis inhibitors and would also identify several other endocrine activities. Because of the difficulties in assessing cytotoxicity and the high false positive/negative rates, the ovary and testis explant assays are not useful as routine screening procedures for detecting steroid biosynthesis inhibitors; however, they may have utility in confirming in vivo findings.

Cucurbitane-type compounds from Hemsleya carnosiflora antagonize ecdysteroid action in the Drosophila melanogaster BII cell line.

The ecdysteroid agonist and antagonist activities of 3 cucurbitanes, 2 cucurbitane glycosides and 2 cucurbitacins isolated from Hemsleya carnosiflora (Cucurbitaceae) have been determined in the Drosophila melanogaster BII bioassay. Carnosiflogenins A and C and carnosiflosides II and VI possess antagonistic activity. Carnosiflogenin A was also found to induce the formation of spindle-shaped cells with high frequency in both the agonist and antagonist assays. At 10(-3) M, carnosiflogenins B and C were cytotoxic, 23,24-Dihydrocucurbitacin F and 25-acetoxy-23,24-dihydrocucurbitacin F are also antagonistic at high concentrations. The concentration dependencies of the antagonistic activities of these two cucurbitacins, carnosiflosides II and VI and carnosiflogenin C are presented. The biological and ecological significance of these results are discussed in relationship to the concentrations present in the rhizomes of H. carnosiflora.

The mechanism of action of steroid antagonists: insights from crystallographic studies.

Examination of the structures of compounds having high affinity for estrogen, progestin, mineralocorticoid and glucocorticoid receptors strongly suggests that receptor binding is primarily the result of a tight association between the receptor and the steroidal A-ring. High affinity binding to the estrogen receptor appears to be dependent upon the presence of a phenolic ring in the substrate. An inverted 1 beta, 2 alpha conformation of the 4-ene-3-one A-ring appears to be most conductive to high affinity binding to the progesterone receptor. Binding to the mineralocorticoid receptor appears to be correlated to a complementary fit between amino acids of the receptor site and a flat 4-en-3-one A-ring similar to that imposed upon aldosterone by the 11,18-epoxide formation. The glucocorticoid receptor appears to prefer a 4-en-3-one A-ring that is bowed toward the alpha-face as is the case in structures having a 9 alpha-fluoro substituent or additional unsaturation at C(1)-C(2). The binding of androgens to their receptor differs in appearing to have an essential dependence upon functional groups at the A- and D-ring end of the steroid. With the exception of the androgens, the data suggest that specific interactions between the steroid B-, C- and D-rings and the receptor play at best a minor role in receptor binding but are the most important factor in determining agonist versus antagonist behavior subsequent to binding. Antagonists that compete for a steroid receptor site may be expected to have the A-ring composition and conformation necessary for receptor binding but lack the 11 beta-OH and the D-ring conformational features and functional groups that induce or stabilize subsequent receptor functions. Antagonists might also be compounds with A-ring conformations appropriate for binding but other structural features that interfere with subsequent receptor functions essential to activity.