The dynamic character of the BCL2 promoter i-motif provides a mechanism for modulation of gene expression by compounds that bind selectively to the alternative DNA hairpin structure.

It is generally accepted that DNA predominantly exists in duplex form in cells. However, under torsional stress imposed by active transcription, DNA can assume nonduplex structures. The BCL2 promoter region forms two different secondary DNA structures on opposite strands called the G-quadruplex and the i-motif. The i-motif is a highly dynamic structure that exists in equilibrium with a flexible hairpin species. Here we identify a pregnanol derivative and a class of piperidine derivatives that differentially modulate gene expression by stabilizing either the i-motif or the flexible hairpin species. Stabilization of the i-motif structure results in significant upregulation of the BCL2 gene and associated protein expression; in contrast, stabilization of the flexible hairpin species lowers BCL2 levels. The BCL2 levels reduced by the hairpin-binding compound led to chemosensitization to etoposide in both in vitro and in vivo models. Furthermore, we show antagonism between the two classes of compounds in solution and in cells. For the first time, our results demonstrate the principle of small molecule targeting of i-motif structures in vitro and in vivo to modulate gene expression.

Characterization of in vitro pharmacokinetic properties of hoodigogenin A from Hoodia gordonii.

This study was aimed to predict the pharmacokinetic properties of hoodigogenin A, which is the aglycone of the oxypregnane steroidal glycoside P57AS3 (P57) isolated from Hoodia gordonii. A series of in vitro assays was used to predict its gastric, intestinal and metabolic stability, intestinal and blood brain barrier (BBB) transport, protein binding and interaction with major drug metabolising enzymes. In the simulated gastric fluid, hoodigogenin A was stable (2 % degradation in 60 minutes) whereas P57 was unstable (45 % degradation in 30 minutes). In simulated intestinal fluid, P57 was degraded to an extent of 8 % in 180 minutes, while hoodigogenin A was stable. Hoodigogenin A was efficiently transported by passive diffusion across Caco-2 and MDR1-MDCK monolayers with P(app) values in the range of 32 x 10(-6) cm/sec and 22 x 10(-6) cm/sec, respectively. The compound was metabolically unstable in human liver microsomes and S9 fractions with a CL' (int) of 71 and 120 mL/min/kg, respectively and was bound to the plasma proteins to an extent of 92 %. The compound strongly inhibited CYP3A4 activity (IC(50) 3 microM), indicating a possibility of drug-herb/botanical interactions when products containing H. gordonii are used simultaneously with other botanicals/herbs/drugs.

Growth inhibition and ultrastructural alterations induced by Delta24(25)-sterol methyltransferase inhibitors in Candida spp. isolates, including non-albicans organisms.

BACKGROUND: Although Candida species are commensal microorganisms, they can cause many invasive fungal infections. In addition, antifungal resistance can contribute to failure of treatment.The purpose of this study was to evaluate the antifungal activity of inhibitors of Delta24(25)-sterol methyltransferase (24-SMTI), 20-piperidin-2-yl-5alpha-pregnan-3beta-20(R)-diol (AZA), and 24(R,S),25-epiminolanosterol (EIL), against clinical isolates of Candida spp., analysing the ultrastructural changes. RESULTS: AZA and EIL were found to be potent growth inhibitors of Candida spp. isolates. The median MIC50 was 0.5 microg.ml-1 for AZA and 2 microg.ml-1 for EIL, and the MIC90 was 2 microg.ml-1 for both compounds. All strains used in this study were susceptible to amphotericin B; however, some isolates were fluconazole- and itraconazole-resistant. Most of the azole-resistant isolates were Candida non-albicans (CNA) species, but several of them, such as C. guilliermondii, C. zeylanoides, and C. lipolytica, were susceptible to 24-SMTI, indicating a lack of cross-resistance. Reference strain C. krusei (ATCC 6258, FLC-resistant) was consistently susceptible to AZA, although not to EIL. The fungicidal activity of 24-SMTI was particularly high against CNA isolates. Treatment with sub-inhibitory concentrations of AZA and EIL induced several ultrastructural alterations, including changes in the cell-wall shape and thickness, a pronounced disconnection between the cell wall and cytoplasm with an electron-lucent zone between them, mitochondrial swelling, and the presence of electron-dense vacuoles. Fluorescence microscopy analyses indicated an accumulation of lipid bodies and alterations in the cell cycle of the yeasts. The selectivity of 24-SMTI for fungal cells versus mammalian cells was assessed by the sulforhodamine B viability assay. CONCLUSION: Taken together, these results suggest that inhibition of 24-SMT may be a novel approach to control Candida spp. infections, including those caused by azole-resistant strains.

The effect of the neuroactive steroid 5beta-pregnane-3beta, 20(R)-diol on the time course of GABA evoked currents is different to that of pregnenolone sulphate.

The endogenous progesterone metabolite allopregnanolone has a number of properties including anesthetic, sedative, antiepileptic, anxiolytic, impaired memory function and negative mood symptoms. Allopregnanolone is a potent positive GABA(A) receptor function modulators. In contrast, 3beta-hydroxy-steroids (3beta-steroids) usually modulate the GABA(A) receptor negatively. They have attracted some interest for their possible use as therapeutic agents that could counteract the negative symptoms induced by allopregnanolone. Two hypotheses for the action of 3beta-steroids have been proposed: 1) 3beta-steroids act in a similar way to pregnenolone sulphate, which non-competitively reduces GABA(A) receptor activity. 2) 3beta-steroids specifically antagonize the effect of allopregnanolone. We have therefore tried to clarify this issue by comparing the effect of pregnenolone sulphate and 5beta-pregnane-3beta, 20(R)-diol on the GABA-evoked currents by the patch clamp technique on neurons from the medial preoptic nucleus. Both pregnenolone sulphate and 5beta-pregnane-3beta, 20(R)-diol increase the desensitization rate of the current response evoked by a 2 s GABA application. However, their effects on other parameters of the GABA evoked currents differed in degree and sometimes even in direction. The actions of pregnenolone sulphate and 5beta-pregnane-3beta, 20(R)-diol were not altered in the presence of allopregnanolone, which indicates that they do not directly interact with allopregnanolone. In addition, when 5beta-pregnane-3beta, 20(R)-diol was tested on spontaneous inhibitory postsynaptic currents (sIPSCs), it dramatically reduced the allopregnanolone-induced prolongation of the decay time constant but it had no effect on the decay under control conditions. In conclusion, the effect of 5beta-pregnane-3beta, 20(R)-diol on GABA-evoked currents is different to that of pregnenolone sulphate in medial preoptic nucleus neurons.

Neurosteroids 3beta, 20 (R/S)-pregnandiols decrease offset rate of the GABA-site activation at the recombinant GABA A receptor.

Neurosteroids directly modulate ligand gated ion channels such as GABA A receptors. Two such molecules, 3beta-OH A-ring reduced pregnane steroids and pregnenolone sulfate (PS), inhibit recombinant GABA A receptor. Using a two-electrode voltage-clamp technique, we compared the effect of 5alpha-pregnan-3beta,20(S)-diol (UC1019), 5beta-pregnan-3beta, 20(R)-diol (UC1020) and PS on the activation onset and offset times of the recombinant GABA A receptor (rat alpha1beta2gamma2L) in Xenopus oocytes. Rapid solution changes allowed the kinetic analysis of GABA-evoked currents. Steroids were co-applied with 30 microM GABA for 10 s, followed by a 80 s washout period. PS (> ir =0.3 microM) moderately increased the slow onset rate (k(on-S)) of GABA-response. PS had no significant effects on the fast onset rate (k(on-F)). UC1019 and UC1020 decreased the k(on-S) of the GABA-response in a concentration-dependent manner with no significant effects on the k(on-F). Like PS, UC1019 and UC1020 decreased the slow offset rates (k(off-S)). In addition, PS increased the fast offset rate (k(off-F)) in a concentration-dependent manner, while UC1019 and UC1020 decreased k(off-F). The EC50 of PS to increase k(off-F) was calculated as 0.47+/-0.1 microM. The corresponding IC50 values of UC1019 and UC1020 to decrease k(off-F) were 5.0+/-0.5 microM and 8.4+/-0.9 microM, respectively. These results suggest differential actions of PS and 3beta, 20(R/S)-pregnandiols on the offset time course of GABA-site activation.

Neurosteroid modulation of recombinant rat alpha5beta2gamma2L and alpha1beta2gamma2L GABA(A) receptors in Xenopus oocyte.

GABA(A) receptors containing alpha(5)-subunit have an important role in cognitive function. As the agonistic effect of 3alpha-hydroxy ring-A reduced steroids depends on subunit combinations of the GABA(A) receptor, the antagonistic effect of pregnenolone sulfate and 3beta-hydroxypregnane steroids may vary between alpha(5)-subunit and alpha(1)-subunit containing receptors. We investigated the effect of agonist and antagonist steroids in the recombinant rat alpha(1)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptors expressed in Xenopus oocytes using a two electrodes voltage-clamp technique. We did not find any significant difference in potency and efficacy of GABA response between alpha(1)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptors. Compared to the alpha(1)beta(2)gamma(2L) receptor, a significantly lower degree of desensitization was observed in the alpha(5)beta(2)gamma(2L) receptor. In addition, the potencies of 3alpha-OH-5alpha-pregnan-20-one (3alpha5alphaP), 5alpha-pregnan-3alpha,21-diol-20-one (3alpha5alphaTHDOC) and 5alpha-androstane-3alpha,17beta-diol (3alpha5alphaADL) to enhance GABA response were significantly higher in the alpha(5)beta(2)gamma(2L) receptor, whereas their efficacies remained unchanged between two receptors. In either receptor, the efficacy of 3alpha5alphaTHDOC was significantly higher than 3alpha5alphaP and 3alpha5alphaADL. The efficacies of 5beta-pregnan-3beta,21-diol-20-one(UC1015) and 5alpha-pregnan-3beta,20alpha-diol(UC1019) to inhibit 30 microM GABA response, and the efficacies of 3beta-OH-5beta-pregnan-20-one (UC1014) and 5beta-pregnan-3beta, 20beta-diol (UC1020) to inhibit 3 microM 3alpha5alphaTHDOC+3 microM GABA response were higher in the alpha(5)beta(2)gamma(2L) receptor compared to the alpha(1)beta(2)gamma(2L) receptor. The potencies of pregnenolone sulfate and 3beta-hydroxypregnane steroids to inhibit the GABA response and the 3alpha5alphaTHDOC+GABA response did not vary between two receptors. Interestingly, the potencies and efficacies of pregnenolone sulfate and 3beta-hydroxypregnane steroids to inhibit the GABA response were positively correlated to their potencies and efficacies to inhibit the 3alpha5alphaTHDOC+GABA response. Results from the current study revealed a different modulation pattern by neurosteroids between the alpha(1)beta(2)gamma(2L) and alpha(5)beta(2)gamma(2L) receptor.

3beta-20beta-dihydroxy-5alpha-pregnane (UC1011) antagonism of the GABA potentiation and the learning impairment induced in rats by allopregnanolone.

Allopregnanolone is a progesterone metabolite and GABA-A receptor modulator with benzodiazepine like effects, including decreased learning and memory. In vitro 3beta-hydroxypregnane steroids antagonize allopregnanolone-induced effects, but no antagonism has been shown in vivo. Our purpose was to evaluate 3beta-20beta-dihydroxy-5alpha-pregnane (UC1011) as a blocker of allopregnanolone-induced effects in vivo and in vitro in rats. We tested adult male Wistar rats in the Morris water maze 8 min after daily injections (i.v.) of allopregnanolone 2 mg/kg (n = 21); allopregnanolone : UC1011 2 : 6 (n = 7), 2 : 8 (n = 7), 2 : 20 (n = 14) mg/kg; UC1011 20 mg/kg (n = 14); or vehicle (10% 2-hydroxypropyl-beta-cyclodextrin, n = 4). Studies of chloride ion uptake into cortical and hippocampal membrane preparations were performed. The latency to find the hidden platform was still high in the allopregnanolone-injected group on day 6. Day 3-6 rats injected with allopregnanolone and UC1011 (2 : 20 mg/kg) had lower latency (P < 0.05), compared to the allopregnanolone-injected group. The group that only received UC1011 learned the location of the platform as fast as the controls. There was no significant difference in swim speed between groups. The time spent swimming close to the pool wall was in the allopregnanolone : UC1011 group (2 : 20 mg/kg) significantly decreased (P < 0.05, day 3-6), compared to the allopregnanolone-injected group. The increased chloride ion uptake induced by increasing dosage of allopregnanolone in the presence of 10 micro m GABA was significantly decreased with UC1011 (P < 0.01), in both cortical and hippocampal homogenates. In conclusion, UC1011 can via antagonism at the GABA-A receptor reduce the negative allopregnanolone effect on learning in the water maze.

Azasterols as inhibitors of sterol 24-methyltransferase in Leishmania species and Trypanosoma cruzi.

This paper describes the synthesis of some novel azasterols based on (20R,22xi)-5alpha-pregnan-20-(piperidin-2-yl)-3beta,20-diol. These compounds are potential inhibitors of the enzyme sterol 24-methyltransferase (24-SMT), which is a vital enzyme in the biosynthesis of ergosterol and related 24-alkyl sterols. Structure-activity studies were undertaken to understand the important features for activity against the enzyme, with the aim of increasing activity and selectivity. The compounds were evaluated for inhibition of recombinant Leishmania major 24-SMT and the effect of compounds on sterol composition and parasite proliferation. Essentially, compounds which showed good activity against the recombinant enzyme had a significant effect on the sterol composition and growth of parasites. The activity of compounds was found to be related to the basicity and stereochemical location of the nitrogen. Also, presence of an unprotected 3beta-OH seemed to be important for activity. However, some azasterols which were not good inhibitors of 24-SMT also showed antiproliferative activity, suggesting that there may be other modes of actions of these compounds.

Studies of molecular structure parameters of 20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol and its N-methyl derivative: two inhibitors of Delta24(25) sterol methyl transferase and Delta24(24') sterol methyl reductase of Trypanosoma cruzi.

Molecular structural parameters of two potential drugs against Trypanosoma cruzi epimastigotes, 20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol (1) and 20-N-methylpiperidin-2-yl-5alpha-pregnan-3beta, 20-diol (2) were studied using a combination of a stereoselective synthetic route, spectroscopic characterization and single-crystal X-ray analysis. Both compounds were synthesized with an R configuration at C20. This chirality is a consequence of the stereoselectivity observed during the formation of the intermediate 20-pyridin-2-yl-5alpha-pregnan-3beta,20R-diol (4). NMR data indicated that the six-membered aza ring of (2) is conformationally more restrained, in CDCl3 solution, than (1). X-ray studies showed that maximum deviations among structural molecular parameters of (1) and (2) correspond to torsion angles along the C20-C22 bonds, leading to a different relative orientation of the N atom; a critical structural parameter for the binding properties of aza-sterols to Delta(24(25)) sterol methyl transferase. Cremer-Pople parameters of the five-membered rings of (1) and (2) lie in the observed range for a family of tetracyclic fused ring systems retrieved from the CSD. The phi2 parameter of (1) lies just on the mean of the family, while phi2 of (2) deviates significantly towards the lower limit.

Steroid structure and pharmacological properties determine the anti-amnesic effects of pregnenolone sulphate in the passive avoidance task in rats.

Pregnenolone sulphate (PREGS) has generated interest as one of the most potent memory-enhancing neurosteroids to be examined in rodent learning studies, with particular importance in the ageing process. The mechanism by which this endogenous steroid enhances memory formation is hypothesized to involve actions on glutamatergic and GABAergic systems. This hypothesis stems from findings that PREGS is a potent positive modulator of N-methyl-d-aspartate receptors (NMDARs) and a negative modulator of gamma-aminobutyric acid(A) receptors (GABA(A)Rs). Moreover, PREGS is able to reverse the amnesic-like effects of NMDAR and GABA(A)R ligands. To investigate this hypothesis, the present study in rats examined the memory-altering abilities of structural analogs of PREGS, which differ in their modulation of NMDAR and/or GABA(A)R function. The analogs tested were: 11-ketopregnenolone sulphate (an agent that is inactive at GABA(A)Rs and NMDARs), epipregnanolone ([3beta-hydroxy-5beta-pregnan-20-one] sulphate, an inhibitor of both GABA(A)Rs and NMDARs), and a newly synthesized (-) PREGS enantiomer (which is identical to PREGS in effects on GABA(A)Rs and NMDARs). The memory-enhancing effects of PREGS and its analogs were tested in the passive avoidance task using the model of scopolamine-induced amnesia. Both PREGS and its (-) enantiomer blocked the effects of scopolamine. The results show that, unlike PREGS, 11-ketopregnenolone sulphate and epipregnanolone sulphate failed to block the effect of scopolamine, suggesting that altering the modulation of NMDA receptors diminishes the memory-enhancing effects of PREGS. Moreover, enantioselectivity was demonstrated by the ability of natural PREGS to be an order of magnitude more effective than its synthetic enantiomer in reversing scopolamine-induced amnesia. These results identify a novel neuropharmacological site for the modulation of memory processes by neuroactive steroids.

Anxiolytic properties of endogenously occurring pregnanediols in two rodent models of anxiety.

Certain endogenously occurring 3 alpha-hydroxylated, 5-reduced pregnane steroids act at a specific site on the GABAA receptor complex (GRC) to modulate the effects of GABA at its receptor. Modulators that potentiate GABA at the GABAA receptor often possess anxiolytic properties. The anxiolytic potential of four 5-reduced, 3 alpha, 20-pregnanediols, differing only in the stereochemical orientation of the steroid A-ring and the 20-hydroxyl group, were tested in the Vogel test following intracerebroventricular (ICV) administration. The effects of these pregnanediols were compared to those of their 20-ketone analogues, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) and 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha, 5 beta-P). All four pregnanediols tested significantly enhanced punished drinking at doses ranging from 10 to 60 micrograms. The rank order of potency based on the minimum effective dose (MED) observed was 5 alpha-pregnan-3 alpha,20 alpha-diol = 5 beta-pregnan-3 alpha,20 alpha-diol > 5 beta-pregnan-3 alpha,20 beta-diol > 5 alpha-pregnan-3 alpha, 20 beta-diol. 3 alpha,5 beta-P and 3 alpha,5 alpha-P enhanced punished responding when administered at 2.5 and 5 micrograms, respectively. 3 beta,5 alpha-P which is inactive at the GRC was also inactive (up to 100 micrograms) in the Vogel test. The benzodiazepine control diazepam was efficacious when administered at 2.5 micrograms. 5 alpha-Pregnan-3 alpha,20 alpha-diol was further tested in the mouse elevated plus-maze model following systemic administration where it was found to be active in a dose range of 10-40 mg/kg IP. These results raise the possibility that in addition to 3 alpha,5 alpha-P and 3 alpha,5 beta-P, some of their endogenously occurring pregnanediol metabolites may also influence physiological processes related to anxiety via the GRC.

Modulation of human recombinant GABAA receptors by pregnanediols.

Utilising two point voltage-clamp techniques on Xenopus laevis oocytes expressing human (alpha 1 beta 1 gamma 2L) recombinant GABAA receptors, the GABA modulatory actions of six naturally occurring neurosteroids have been determined and compared with those of known positive allosteric modulators. The anaesthetic steroids 5 alpha- and 5 beta-pregnan-3 alpha-ol-20-one produced a concentration-dependent enhancement of the GABA-evoked current. The maximal enhancement of the agonist-induced response produced by these steroids was intermediate between that of pentobarbitone and diazepam, but much greater than that caused by bretazenil. For both the 5 alpha and 5 beta steroid a reduction of the 20 ketone group to form either the corresponding 20 alpha or 20 beta hydroxy steroid produced, in all cases, a reduction in potency and a decrease in the maximal effect. The relationship of steroid structure to these two parameters is considered. The influence of the alpha subtype (alpha x beta 1 gamma 2L, where x = 1, 2 or 3) for the behaviourally active 5 alpha-pregnan-3 alpha,20 alpha-diol is also determined. Although the maximal effect of the steroid is not influenced by the alpha subtype, the alpha 2-containing receptor exhibits a modest decrease (approximately 6-fold) in potency compared to alpha 1- and alpha 3-containing receptors. The results described here are discussed in relation to the distinct behavioural actions of the neurosteroids.

Modulation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in the equine placenta by pregnenolone and progesterone metabolites.

The purpose of this study was to measure 3beta-HSD activity in the equine placenta and to assess the effect of fetal and maternal blood plasma progestagens on 3beta-HSD activity was measured in 8 late gestation (collected by caesarian section at 250 to 320 days) and 7 term (collected by caesarian section at 250 to 320 days) and 7 term (collected at birth) equine placentae using a tritium release assay with [3alpha-3H] pregnenolone as substrate. Mean +/- s.d. Km(app) and Vmax for term placentae were in general higher than for late gestation placentae (0.129 +/- 0.217 micromol/l and 23.85 +/- 9.1 nmol/mg/h respectively vs. 0.016 +/- 0.048 micromol/l and 17.36 +/- 20.9 nmol/mg/h) but there was no statistical difference between them. Inhibition studies were performed on 3 term placentae and 3 late gestation ones. Steroid concentrations used for inhibition studies were close to blood plasma concentrations (0.5 to 2 micromol/l). 3beta-hydroxy compounds (5alpha-pregnene-3beta, 20alpha-diol and 3beta-hydroxy-5alpha-pregnan-20-one) showed noncompetitive or mixed inhibition. Mean Ki(app) of 0.7 micromol/l. Inhibition was competitive with 20alpha-hydroxy-5alpha-pregnan-3-one with a mean Ki(app) of 0.1 micromol/l.

Selective actions of certain neuroactive pregnanediols at the gamma-aminobutyric acid type A receptor complex in rat brain.

Certain endogenous pregnanediols (5 alpha-pregnan-3 alpha,20 alpha-diol and 5 beta-pregnan-3 alpha,20 beta-diol) were observed to have limited efficacy as allosteric modulators of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) and [3H]flunitrazepam binding to sites on the gamma-aminobutyric acid (GABA)A receptor complex in rat brain. In contrast, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha,5 beta-P) have full efficacy. Moreover, 3 alpha,5 beta-P but not 3 alpha,5 alpha-P recognizes high (nanomolar) and low (micromolar) affinity neuroactive steroid sites in these allosteric modulatory assays. The concentration-response curve for 3 alpha,5 alpha-P modulation of [35S]TBPS binding was shifted rightward in the presence of these pregnanediols and GABA. The maximum shift produced by these pregnanediols never exceeded the concentration-response curve obtained with 3 alpha,5 alpha-P alone in the absence of GABA. Additionally, neither 5 alpha-pregnan-3 alpha,20 alpha-diol nor 5 beta-pregnan-3 alpha,20 beta-diol had any effect on the site recognized by 3 alpha,5 alpha-P in the absence of GABA. The difference in the affinities of the two apparent sites (29 nM versus 152 nM in the presence and absence of GABA, respectively) recognized by 3 alpha,5 alpha-P is only approximately 5-fold. In contrast, the difference between the high (30 nM) and low (7 microM) affinity sites discriminated by 3 alpha,5 beta-P is > 200-fold. Thus, the selective interaction between the high affinity site recognized by 3 alpha,5 beta-P and these pregnanediols can be clearly observed. A saturating concentration of 5 beta-pregnan-3 alpha,20 beta-diol selectively eliminated the high affinity component recognized by 3 alpha,5 beta-P, whereas 5 alpha-pregnan-3 alpha,20 alpha-diol did not completely abolish the high affinity site. 5 alpha-Pregnan-3 alpha,20 alpha-diol recognized only a portion of the high affinity sites discriminated by 3 alpha,5 beta-P, relative to 5 beta-pregnan-3 alpha,20 beta-diol, whereas the two pregnanediols recognized a similar population of sites mediating 3 alpha,5 alpha-P inhibition of [35S]TBPS binding. Collectively, these studies provide evidence that the limited efficacy of certain pregnanediols as allosteric modulators of [35S]TBPS binding may be explained in part by selectivity for the high affinity site recognized by 3 alpha,5 beta-P.(ABSTRACT TRUNCATED AT 400 WORDS)

Alterations of the regiospecificity of progesterone metabolism by the mutagenesis of two key amino acid residues in rabbit cytochrome P450 2C3v.

A Ser/Thr difference at position 364 underlies a phenotypic difference between naturally occurring variants of microsomal cytochrome P450 2C3 in their capacity to catalyze the 6 beta-hydroxylation of progesterone, as well as in their sensitivity to the inhibitor 16 alpha-methyl-progesterone. Position 364 of P450 2C3 maps to a substrate contacting domain suggested by models for mammalian P450 enzymes based on the structure of P450,101. In this study, Thr-364 of P450 2C3v, a progesterone 6 beta- and 16 alpha-hydroxylase, was replaced by Gly, Asp, Asn, Val, Leu, or Ile. The latter three amino acids did not alter the regiospecificity of P450 2C3v, whereas the Gly, Asp, and Asn substitutions each produced enzymes with properties that correspond closely to the Ser mutant that catalyzes 16 alpha-hydroxylation but not 6 beta-hydroxylation. The former are distinguished from the latter amino acids by their greater hydrophobicity and size. In contrast, the 16 alpha-hydroxylase activity could be greatly diminished by the introduction of an alanine replacement for Val-113. This mutation conferred progesterone 21- and 17 alpha-hydroxylase activity to P450 2C3v at the expense of 16 alpha-hydroxylase activity, leaving the 6 beta-hydroxylase activity largely unaffected. Compound mutants displayed the additive effects of the two mutations. These results are consistent with two distinct orientations for the binding of progesterone to P450 2C3v, resulting in 6 beta- and 16 alpha-hydroxylation, respectively. The binding of progesterone in these two orientations can be modulated relatively independently by modifications of the two key amino acid residues at 113 and 364.

Complex interactions between the steroid derivative RU 5135 and the GABAA-receptor complex.

The modulation of [35S]t-butylbicyclophosporothionate ([35S]TBPS) binding was used to evaluate the actions of the steroid derivative RU 5135 at the gamma-aminobutyric acidA (GABAA) receptor complex. The inhibition of [35S]TBPS binding by GABA in the presence of various concentrations of RU 5135 was consistent with the hypothesis that RU 5135 is a competitive antagonist at the GABAA receptor. Despite common structural features (i.e., 3 alpha-hydroxylated, 5 beta-reduced A ring) with GABAA receptor-active neurosteroids, RU 5135 did not appear to be competitive at the putative steroid site on the GABAA receptor-active, as demonstrated by Schild analysis of 5 alpha-pregnane-3 alpha-ol-20-one (3 alpha,5 alpha-P) modulation of [35S]TBPS binding in the presence of different concentrations of RU 5135. On the other hand, the reduced potency of 3 alpha,5 alpha-P as an inhibitor of [35S]TBPS binding in the presence of RU 5135, as well as blockade of 5 alpha-pregnane-3 alpha-20 alpha-diol (5 alpha-pregnanediol) inhibition of [35S]TBPS binding by RU 5135 provide further support for the GABAA receptor antagonist properties of RU 5135. Moreover, this amidine steroid was able to partially inhibit [35S]TBPS binding independent of GABA with nanomolar potency; yet the mechanism by which this occurs remains to be determined.

Gamma-aminobutyric acidA receptor complexes in rat frontal cortex and spinal cord show differential responses to steroid modulation.

Regional differences in neuroactive steroid modulation of the gamma-aminobutyric acidA receptor-chloride ionophore complex (GBRC), as measured by t-butylbicyclophosphoro[35S]thionate ([35S] TBPS) binding and 36Cl- uptake, were demonstrated in rat spinal cord versus frontal cortex. The rank order of potencies of a series of 5 alpha- and 5 beta-reduced isomers of 3 alpha-hydroxylated steroids against [35S]TBPS binding were different between regions. The differences in rank order of potencies imply the possible existence of heterogeneous populations of GBRC-coupled steroid recognition sites. The relative potencies of selected 5 alpha- and 5 beta-reduced isomers as potentiators of 36Cl- uptake paralleled their potencies as inhibitors of [35S]TBPS binding. Differential sensitivity of the steroid recognition site to the allosteric influence of gamma-aminobutyric acid was also demonstrated. It appears that regionally specific responses to GBRC-active steroids do occur, although the functional consequences of these effects await evaluation in appropriate in vivo models.

Ipsilateral and contralateral effects on cutaneous reflexes in a back muscle of the female rat: modulation by steroids relevant for reproductive behavior.

1. Multiunit EMG recordings of cutaneous reflexes--evoked in the back extensor, lateral longissimus (LL), by bilateral stimulation of nerves to dorsal flank skin--were studied in ovariectomized female rats with and without estrogen pretreatment. 2. Poststimulus time (PST) histograms of data from rats with and without estrogen pretreatment show that the axial EMG response (10-30 ms) to ipsilateral (ipsi) flank skin nerve stimulation is significantly shorter in latency (1.4 ms) and 67% larger than the response to contralateral (contra) flank skin nerve stimulation recorded at the same site (n = 29 pairs). 3. When late EMG responses were also evoked at 50-120 ms in 37% of ipsi and 29% of contra cutaneous reflexes, the incidence of multiunit activity in the late peak was significantly greater in rats pretreated with silastics containing 100% estradiol (E) compared with 10% E or cholesterol controls. 4. When an ipsi cutaneous reflex in LL was conditioned by a stimulus to the contra flank skin nerve at a condition-test interval of 30 ms (C-T 30 ms), the average number of discharges in the early peak of the histogram was double that in the histogram obtained from the unconditioned ipsi reflex, independent of estrogen pretreatment. 5. In 12 out of 19 cases in which a contra cutaneous reflex was conditioned by a stimulus to the ipsi flank skin nerve (C-T 30 ms), the number of discharges in the early peak of the histogram was less than that in the early peak of the histogram derived from the unconditioned contra response, independent of estrogen pretreatment. 6. Intravenous injections of progesterone (P) or its metabolite 5 alpha-pregnane-3 alpha-ol-20-one (tetrahydraprogesterone, THP) decreased the magnitude of the early peak of the ipsi cutaneous reflex and the contra cutaneous reflex in LL, independent of estrogen pretreatment. At the same time, it did not reduce the magnitude of the early peak evoked by either ipsi or contra nerves after conditioning from the other side at C-T 30 ms. 7. As a consequence, the percentage facilitation of ipsi cutaneous reflexes by contra cutaneous conditioning was significantly increased after P or THP. This suggests that these steroids can selectively enhance behaviors involving bilateral inputs. 8. An unchanged axial motoneuron pool output to bilateral cutaneous inputs after P and THP, in spite of reduced motoneuron responses to cutaneous inputs from each side of the body, implies an increased gain in the reflex circuit.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterotropic cooperativity between putative recognition sites for progesterone metabolites and the atypical benzodiazepine Ro 5-4864.

The binding of the cage convulsant t-butylbicyclophosphorothionate (TBPS) and 36Cl- uptake by synaptoneurosomes were used to test the ability of progesterone metabolites to modulate allosterically the Ro 5-4864 (4'-chlorodiazepam) binding site that is functionally coupled to the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex (GBRC) in rat brain. Dose-dependent enhancement of [35S]TBPS binding by Ro 5-4864 occurs in rat cerebral cortex in the presence of the progesterone metabolites 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) and 5 alpha-pregnan-3 alpha, 20 alpha-diol (pregnanediol). The pregnanediol effect is completely GABA dependent, whereas that of 3 alpha-OH-DHP is not. Conversely, Ro 5-4864 opposed the action of 3 alpha-OH-DHP by increasing the IC50 for 3 alpha-OH-DHP inhibition of [35S]TBPS binding. In cortical synaptoneurosomes, Ro 5-4864 antagonized both 3 alpha-OH-DHP and pregnanediol enhancement of GABA-stimulated 36Cl- uptake. In both binding and functional studies, pregnanediol showed limited efficacy relative to 3 alpha-OH-DHP, as previously reported. These findings provide the initial evidence that the GBRC-linked Ro 5-4864 binding site is allosterically coupled to the putative progesterone metabolite recognition site and confirm the GABA-mimetic properties of 3 alpha-OH-DHP and pregnanediol.

Enhancement of [3H]muscimol binding to brain synaptic membranes by progesterone and related pregnanes.

Progesterone (a delta 4-3 keto-pregnane) as well as a series of pregnanes enhanced [3H]muscimol binding to rat cerebral cortex membranes. This effect was increased by preincubation in the presence of the drugs, progesterone being effective only after a 30 min preincubation. The effect of progesterone was dose-dependent from 1 nM to 100 microM reaching a maximum of 140% over control. Its 20 alpha and 20 beta reduced metabolites (20 alpha- and 20 beta-OH-4-pregnen-3-one) had no effect. Ring A reduction in either the 5 alpha or 5 beta position resulted in steroids (5 alpha- and 5 beta-pregnane-3,20-di-one) producing moderate but consistent facilitation of binding (30-40% increase at 10 microM). The presence of a hydroxyl group in C3 had variable results in the potency of pregnanes to facilitate muscimol binding. Pregnanes with a 3 beta-OH group showed weaker activity than those having a 3 alpha-OH group, 5 alpha-pregnan-3 alpha-ol-20-one being the most potent (100% stimulation). Pregnenolone, a delta 5-3 beta-OH-pregnane, was only effective at low concentrations (10 nM to 10 microM). Scatchard analysis of [3H]muscimol binding in the presence of progesterone and 5 alpha-pregnan-3 alpha-ol-20-one revealed that the observed effect was due to an increase in binding sites rather than to a change in affinity.