EFFECTS OF ANDROSTENEDIONE, 19-NORETHYNYLTESTOSTERONE, PROGESTERONE, AND 17α-HYDROXYPROGESTERONE UPON THE MANIFESTATION OF SECONDARY SEXUAL CHARACTERS IN THE MEDAKA, ORYZIAS LATIPES.

Effects of androstenedione, 19-norethynyltestosterone, progesterone, and 17α-hydroxyprogesterone upon the manifestation of secondary sexual characters were studied in the orange-red variety of the medaka. The steroids were consecutively administered per os for 15 days. Androstenedione and 19-norethynyltestosterone induced formation of new segments in the fin rays and produced papillar processes on the anal fin of the female. 19-norethynyltestosterone proved to have a strong potency, estimated to be more than 300 times that of androstenedione. Progesterone and 17α-hydroxyprogesterone had neither androgenic nor estrogenic effect on the secondary sexual characters. This is quite in harmony with the fact that they have neither androtermonic nor gynotermonic potency. The administration of these steroids did not impair the fertility of females. The administered females produced offspring by mating with administered males.     

Metabolic pathways for androstanediol formation in immature rat testis microsomes

Metabolic routes from progesterone to androstanediol in washed rat testicular microsomes were investigated, with special emphasis on the importance of 4-ene-3-oxosteroids, as well as the effect of a minimal effective dose of human chorionic gonadotropin on these transformations. Incubation of equimolar concentrations of a mixture of [¹⁴C]progesterone and 17α-hydroxy[³H]progesterone resulted in a large preference of 17α-hydroxyprogesterone over progesterone as substrate for androstanediol formation. Incubation of [³H]progesterone together with [¹⁴C]androstenedione resulted in the inhibition of C-17,20-lyase and in a low ¹⁴C/³H ratio in androstanediol, indicating the preference of progesterone over androstenedione as substrate for androstanediol production. When a mixture of 17α-hydroxyl[³H]progesterone and [¹⁴C]androstenedione was incubated with the microsomes, a more than 8-fold preference of 17α-hydroxyprogesterone as substrate for androstanediol production was found. The minimal dose of human chorionic gonadotropin stimulated testosterone production but inhibited androstanediol formation and effected, in some instances, a change in the metabolic routes. It is concluded that androstanediol is produced preferentially through 17-hydroxylated C-21 steroids, and also, to a lesser extent, through C-19 steroids.     

Inhibition of 17,20(17-hydroxyprogesterone)-lyase by progesterone

¹⁴C-17-Hydroxyprogesterone was incubated with 7000 × g × 20 min supernatants of rat testis homogenates in the presence of various concentrations of ³H-progesterone, both under conditions where metabolism would take place and where it would be prevented. When metabolism was prevented, the ratio of progesterone to 17-hydroxyprogesterone in the microsomal fraction was 3 times that which was added to the incubation medium.Progesterone competitively inhibited 17,20-lyase action on added 17-hydroxyprogesterone but not on 17-hydroxyprogesterone formed from the added progesterone. The rate of formation of 17-hydroxyprogesterone from progesterone, however, was inhibited by added 17-hydroxyprogesterone. The results indicate that there is no free exchange of an intermediate between progesterone and androstenedione with the soluble fraction, either inside or outside the microsomal vesicle. The limited exchange with 17-hydroxyprogesterone in solution probably represents exchange with an enzyme-bound intermediate.     

Pathway of testosterone biosynthesis in the testis of the marmoset Saguinus oedipus

These studies were undertaken to determine the principal pathway of androgen biosynthesis by the testis of the marmoset $\text{Saguinus oedipus}$. Testicular fragments (25 mg) were incubated at 37°C in Krebs-Ringer bicarbonate buffer, pH 7.4, containing pregnenolone-7-³H (3β-hydroxy-5-pregnen-20-one) or progesterone-7-³H. Duplicate fragments were incubated with each substrate for 30 min, one hr, three hr, or five hr, for a total of 16 separate incubations. Metabolites were separated by paper and thin-layer chromatography, with identity established by recrystallization to constant specific activities and ³H/¹⁴C ratios. Pregnenolone was readily metabolized to progesterone, 17α-hydroxyprogesterone, androstenedione (4-androstene-3, 17-dione) and testosterone. Progesterone was converted to 17α-hydroxyprogesterone, androstenedione and testosterone. 17α-hydroxyprogesterone was the predominant metabolite obtained from both substrates at one, three and five hrs of incubation. Neither 17α-hydroxypregnenolone (3β-17-dihydroxy-5-pregnen-20-one) nor dehydroepiandrosterone (3β-hydroxy-5-androsten17-one) was detected in the incubates. These data suggest a predominant Δ⁴ pathway with accumulation of 17α-hydroxyprogesterone in the testis of this primate specie.     

Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS

Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants—terrestrial manatee relatives—were targeted. These included 5α-reduced progestins (5α-pregnane-3,20-dione [5α-DHP] and 3α-hydroxy-5α-pregnan-20-one [5α-P3-OH]) and 17α-hydroxyprogesterone (17α-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20α-hydroxyprogesterone (20α-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5α-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5α-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17α-OHP metabolite was not detected in any tested female. The 20α-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.     

Range of substrates and steroid bioconversion reactions performed by recombinant microorganisms Saccharomyces cerevisiae and Yarrowia lipolytica expressing cytochrome P450c17

The relationship between 17α-hydroxylation and 20-oxidation-reduction of progesterone and some of its derivatives was studied in yeast strains Saccharomyces cerevisiae YEp51α, Yarrowia lipolytica E129A15, and expressing cytochrome P450c17. The key metabolites were found to be 17α-hydroxyprogester-one and 17α,20(α,β)-dihydroxypregn-4-ene-3-ones. The bioconversion pathways of pregn-4-ene-20(α,β)-ol-3-ones were determined. They included cycles of 20-oxidation, 17α-hydroxylation, and stereospecific 20-reduction. The efficiency and kinetic parameters of steroid bioconversion by the recombinant strains were determined. The role of yeast analogs of mammalian steroid dehydrogenases is discussed. It was found that any of the desired derivatives, 17α-hydroxyprogesterone or progesterone 17α,20(α,β)-diols, could be obtained from progesterone. Cholesterol bioconversion yields important metabolites: steroid hormones, the vitamin-D group, and bile acids [1, 2]. Attention to various cytochrome-P450 species participating in the biosynthesis of mammalian steroid hormones is caused by two circumstances: (1) the necessity of detecting structural-function abnorm alities of some of the enzymes of steroid-synthesis that cause human diseases, and (2) the potential of regio-and stereospecific cytochrome P450 species of mammals in chemoenzymatic synthesis of pharmacologically valuable steroids. Concerning the second line of inquiry, the development of transgenic Saccharomyces cerevisiae yeast for the complete synthesis of cortisol by additional expression and elimination of a total of 13 genes was reported [3]. To increase the yield of the target compound, the genes for enzymes performing undesirable steroid modifications were inactivated. These modifications included esterification of pregnenolone [4] and 20α-reduction of 17α-hydroxyprogesterone [5]. A search for analogs of mammalian 20α-hydroxysteroid dehydrogenase (20α-HSD) in the Saccharomyces cerevisiae genome revealed two candidate proteins: Ypr1p (yeast aldo-keto reductase) and Gcy1p (yeast galactose-inducible crystallin-like protein) [3]. Indeed, it was formerly shown that expression of cytochrome P450 from bovine adrenal cortex, performing 17α-hydroxylation and the C17,20-lyase reaction (P450c17) in S. cerevisiae under the control of the GAL10-promoter with the presence of D-galactose as an inducer, was accompanied by the sequential conversion of progesterone to 17α-hydroxyprogesterone and 17α,20(α,β)-dihydroxypregn-4-ene-3-one with a high yield [5].     

Pregnenolone sulphate-independent inhibition of TRPM3 channels by progesterone

Transient Receptor Potential Melastatin 3 (TRPM3) is a widely expressed calcium-permeable non-selective cation channel that is stimulated by high concentrations of nifedipine or by physiological steroids that include pregnenolone sulphate. Here we sought to identify steroids that inhibit TRPM3. Channel activity was studied using calcium-measurement and patch-clamp techniques. Progesterone (0.01-10μM) suppressed TRPM3 activity evoked by pregnenolone sulphate. Progesterone metabolites and 17β-oestradiol were also inhibitory but the effects were relatively small. Dihydrotestosterone was an inhibitor at concentrations higher than 1μM. Corticosteroids lacked effect. Overlay assays indicated that pregnenolone sulphate, progesterone and dihydrotestosterone bound to TRPM3. In contrast to dihydrotestosterone, progesterone inhibited nifedipine-evoked TRPM3 activity or activity in the absence of an exogenous activator, suggesting a pregnenolone sulphate-independent mechanism of action. Dihydrotestosterone, like a non-steroid look-alike compound, acted as a competitive antagonist at the pregnenolone sulphate binding site. Progesterone inhibited endogenous TRPM3 in vascular smooth muscle cells. Relevance of TRPM3 or the progesterone effect to ovarian cells, which have been suggested to express TRPM3, was not identified. The data further define a chemical framework for competition with pregnenolone sulphate at TRPM3 and expand knowledge of steroid interactions with TRPM3, suggesting direct steroid binding and pregnenolone sulphate-independent inhibition by progesterone.     

Progestin metabolism in the rhesus monkey corpus luteum

For the purpose of describing the pathway by which estrogens are synthesized in the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) corpus luteum (CL), CL were obtained during the midluteal phase of the menstrual cycle and fragments incubated with equimolar amounts of [7-³H]pregnenolone plus [4-¹⁴C]progesterone. Metabolites including ³H-progesterone, ³H, ¹⁴C-20α-dihydroprogesterone, ³H, ¹⁴C-17-hydroxyprogesterone, ³H-estrone and ³H-estradiol-17β appeared in the medium during the first 20 minutes of incubation, ³H, ¹⁴C-Androstenedione was not consistently noted until after 60 minutes. Despite the fact that the ¹⁴C/³H-17-hydroxyprogesterone ratio quickly approached a constant value in the medium, ¹⁴C-estrogens were not detected in the medium or tissue fragments suggesting that progesterone was not a principal precursor for estrogen synthesis. As evidenced by the observation that the ¹⁴C/³H-progesterone ratio was significantly higher in luteal fragments than the 17-hydroxyprogesterone ratio, 17-hydroxyprogesterone appeared to be synthesized from pregnenolone both by way of progesterone and by another route which did not include progesterone. C₂₁- and C₁₈-Steroids were more concentrated in tissue fragments after 120 minutes of incubation than in the medium indicating that these steroids were sequestered by luteal tissue.     

Progesterone, 17α-hydroxyprogesterone,androgens and oestrogens in bovine ovarian cysts

The concentration of steroid hormones in cystic follicular fluid was determined in cows with cystic ovaries. There was a significant difference in concentrations of the hormones from the cysts with granulosa cell layers, and the cysts without granulosa cell layers or only 2 to 3 layers with pycnotic nuclei in the granulosa cells (P < 0.01). The cystic follicles that consist of both thecal- and granulosa cell layers contained a low amount of progesterone and high levels of 17α-hydroxyprogesterone, androgens and oestrogens, not different from preovulatory follicles.In contrast, cysts that consist only of thecal cell layers contained a very high amount of progesterone, but very low levels of 17α-hydroxyprogesterone, androgens and oestrogens. That is functionally similar to the bovine corpus luteum which produces high concentrations of progesterone but has no or very low 17α-hydroxylase activity.In conclusion, the cystic follicles without granulosa cells are not capable of secreting 17α-hydroxyprogesterone, androgens and oestrogens, in spite of high levels of progesterone. It may be suggested that in these cysts there is a blockade of 17α-hydroxylase activity.     

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.     

Properties of antisera to progesterone and to 17-hydroxy-progesterone elicited by immunization with the steroids attached to protein through position 7

Antibodies to progesterone (P) and to 17-hydroxyprogesterone (17-OHP) were raised by immunization of rabbits with progesterone-7α-carboxyethyl thioether--bovine serum albumin (P-7—BSA) or with 17-OHP-7α-carboxyethyl thioether--BSA (17-OHP-7--BSA). The antisera produced were of high affinity: K_a towards the homologous hapten was 3. 7 × 10¹⁰ 1./mol for the anti-P serum and 5. 9 × 10⁹ 1/mol for the anti-17-OHP serum. The antiserum to P-7—BSA displayed little or no cross reaction (?= 2%) with the 20α-, 20β- or 5β-dihydro-derivatives of progesterone, moderate cross-reaction with pregnenolone (4%), but considerable cross-reaction with 11-deoxycorticosterone (7%), 5α-dihydro-progesterone (11%) and 17-OHP (15%). The antiserum to 17-OHP-7--BSA showed very little cross-reaction (?= 2%) with progesterone and other steroids lacking a 17α-hydroxyl group, such as pregnenolone or 11-deoxycorticosterone, but reacted significantly with 17α, 21-dihydroxy-4-pregnene-3, 20-dione (8%) and 3β, 17-dihydroxy-5-pregnen-20-one (13%). None of the sera reacted with testosterone, cortisol or estradiol-17β. It appears that conjugation of progesterone to protein through carbon-7 affords antisera comparable in specificity to those raised with 11α-conjugates and superior to those raised with 3-, 6- and 20-conjugates. The antiserum to 17-hydroxyprogesterone described is the first one that specifically recognizes this metabolite.     

Corticoid formation by the monkey fetal adrenal: Evaluation of 17-, 21- and 11-hydroxylase activities

There is indirect evidence that cortisol synthesis in the fetal rhesus monkey adrenal gland is limited at Day 135 of gestation but increases thereafter. This study was conducted to ascertain whether a reduced synthetic capacity is caused by a deficiency in 17-, 21- or 11-hydroxylase activity. For the sake of comparison 11- and 21-hydroxylases were also estimated in adult adrenals. 11-, 21-Hydroxylases were measured in the entire adrenal by the oxidation of NADPH by mitochondria and microsomes, respectively. 17-Hydroxylase was evaluated in outer and inner regions of the fetal gland by the formation of [³H]17-hydroxyprogesterone, -11-deoxycortisol, -cortisol and -androstenedione from [³H]progesterone. The maximum velocity of both the 11- and 21-hydroxylase was similar in fetal and adult glands indicating that corticoid formation in the fetus is not constrained by levels of these enzymes.[³H]Progesterone was extensively metabolized to -17-hydroxyprogesterone, -androstenedione, -11-deoxycortisol and -cortisol by homogenates from both regions of the fetal adrenal. The ratio of [³H]-cortisol to [³H]11-deoxycortisol was consistently higher in incubations of the inner glandular area. Together, these findings indicate that 17-hydroxylase is also active at Day 135 and that the 11-hydroxylase may be more concentrated in the fetal cortex. These data suggest in addition that the restriction in cortisol formation occurs at a step prior to the metabolism of progesterone to cortisol.     

Identification and quantification of C21 and C19 steroids in the haemolymph of Leptinotarsa decemlineata,a phytophagous insect

o-Pentafluorobenzyloxime (OPFB)-heptafluorobutyrylester (HFB)-derivatives were prepared from extracts of haemolymph from last instar larvae of Leptinotarsa decemlineata and subjected to negative ion chemical ionization capillary gas chromatography-mass spectrometry (NCI/GC-MS). Ten C21 and C19 steroids could be positively identified: testosterone, dehydroepiandrosterone, 5α-dihydrotestosterone, 11-ketotestosterone, 11β-hydroxytestosterone, androstenedione, progesterone, 17α-hydroxyprogesterone, pregnenolone and 17α,20β-dihydroprogesterone. No estrogens could be found in these larvae. Radioimmunoassay of chromatographed extracts of haemolymph taken from the larval and pupal stages showed fluctuations in testosterone (and 5α-dihydrotestosterone) titer.     

Steroidal 5α-reductase inhibitors using 4-androstenedione as substrate

The aim of this study was to determine the capacity of some progesterone derivatives, to inhibit the conversion of labeled androstenedione ([(3)H] 4-dione) to [(3)H]dihydrotestosterone ([(3)H]DHT) in prostate nuclear membrane fractions, where the 5α-reductase activity is present. The enzyme 5α-reductase catalyzes the 5α-reduction of 4-dione whereas the 17β-hydroxysteroid dehydrogenase catalyzes the transformation of 4-dione to testosterone or 5α-dione to dihydrotestosterone (DHT). Moreover, we also investigated the role of unlabeled 5α-dione in these pathways. In order to determine the inhibitory effect of different concentrations of the progesterone derivatives in the conversion of [(3)H] 4-dione to [(3)H]DHT, homogenates of human prostate were incubated with [(3)H] 4-dione, NADPH and increasing concentrations of non-labeled 5α-dione. The incubating mixture was extracted and purified using thin layer chromatography. The fraction of the chromatogram corresponding to the standard of DHT was separated and the radioactivity determined. The results showed that the presence of [(3)H] 4-dione plus unlabelled 5α-dione produced similar levels of DHT as compared to [(3)H] 4-dione. On the other hand, the results indicated that 17α-hydroxypregn-4-ene-3,20-dione 5 and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b, were the most potent steroids to inhibit the conversion of [(3)H] 4-dione to [(3)H]DHT, showing IC(50) values of 2 and 1.6?nM, respectively.     

Interaction of prostaglandins with adrenal microsomal cytochrome P-450 in the guinea pig

Studies were carried out to investigate the effects of prostaglandins (PG) $\text{in vitro}$ on adrenal microsomal steroid and drug metabolism in the guinea pig. The addition of PGE₁, PGE₂, PGA₁, PGF_1α or PGF_2α to isolated adrenal microsomes produced typical type I difference spectra. The sizes of the spectra (ΔA_385–420) produced by prostaglandins were smaller than those produced by various steroids including progesterone, 17-hydroxyprogesterone and 11β-hydroxyprogesterone. However, the affinities of prostaglandins and steroids for adrenal microsomal cytochrome P-450, as estimated by the spectral dissociation constants, were similar. Prior addition of prostaglandins to isolated adrenal microsomes did not affect steroid binding to cytochrome P-450 or the rate of steroid 21-hydroxylation. In contrast, prostaglandins inhibited adrenal metabolism of ethylmorphine and diminished the magnitude of the ethylmorphine-induced spectral change in adrenal microsomes. The results indicate that prostaglandins inhibit adrenal drug metabolism by interfering with substrate binding to cytochrome P-450. Since 21-hydroxylation was unaffected by PG, different cytochrome P-450 moieties are probably involved in adrenal drug and steroid metabolism.     

Hydroxysteroid dehydrogenase activity in tissues of two insect species

• 1.1. The metabolism of two tritium labelled vertebrate-type steroids was studied in two insect species, i.e. the fleshfly, Sarcophaga bullata, and the Colorado potato beetle, Leptinotarsa decemlineata.
• 2.2. After injection of [³H]androstenedione into Sarcophaga bullata pharate adults, testosterone (both as free steroid and as conjugate) could be identified as a metabolic product. This indicates the presence of the 17β-hydroxysteroid dehydrogenase (HSD) enzyme in the fleshfly.
• 3.3. Injection of 17α-hydroxy[³H]progesterone into Leptinotarsa decemlineata last instar larvae resulted in the formation of 17α-hydroxy-20α-dihydroprogesterone, 17α-hydroxy-20β-dihydroprogesterone and their conjugates. This indicates the presence of both the 20α-HSD and the 20β-HSD enzyme in Leptinotarsa.
• 4.4. Important conversions in the biosynthetic pathway of steroids in vertebrates, such as the conversion of 17α-hydroxyprogesterone to androgens (Leptinotarsa) and the aromatization of androgens to estrogens (Sarcophaga), were not demonstrated in the metabolic studies.

     

Progesterone transformations by three species ofHumicola

Five isolates belonging to three species of the genusHumicola were tested in this study for their ability to transform progesterone. An oxidative splitting of the side chain of progesterone with the formation of androst-4-ene-3,17-dione, testosterone and testololactone was achieved by all isolates tested ofH. fuscoatra andH. grisea. H. hyalothermophila transformed progesterone to 11α-, 11β-, 17α- and 21-hydroxyprogesterone and a dihydroxyl product (11α, 17α-dihydroxyprogesterone) with the addition of two trihydroxyl products,viz. cortisol and epicortisol. Qualitative and quantitative analysis of the different products obtained when a selective isolate of each species acted on progesterone were conducted. The chromatographic resolution of the mixture products obtained when the selective isolate of each ofH. fuscoatra andH. grisea had acted individually on 1 g progesterone revealed the presence of 25 and 20% unchanged progesterone, 20 and 22% androst-4-ene-3,17-dione, 25 and 23% testosterone and 30 and 35% testololactone, respectively. Seventy-four % of progesterone were bioconverted byH. hyalothermophila into 21-hydroxyprogesterone (6%), 17α-hydroxyprogesterone (5%), 11α-hydroxyprogesterone (11%), 11β-hydroxyprogesterone (12%), 11α,17α-dihydroxyprogesterone (5%), cortisol (21%) and epicortisol (13%). This is the first record of conversion of progesterone to both cortisol and epicortisol byH. hyalothermophila.     

In vitro metabolism of a mixture of [4-C]pregnenolone and [17α-H]pregnenolone by polycystic ovary: Pathways of testosterone biosynthesis

When minced polycystic ovarian tissue was incubated with a mixture of [4-¹⁴C]-pregnenolone and [17α-³H]pregnenolone² and added cofactors for 25, 40 and 60 min, the following metabolites were isolated and characterized: progesterone, 17-hydroxyprogesterone, 17-hydroxypregnenolone, dehydroepiandrosterone, 4-androstenedione, and testosterone; unmetabolized substrates were also recovered. There were increased amounts of progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, and 4-androstenedione formed as the incubation time increased.     

Preconditioning with estradiol abolishes its neuroprotection in cerebellar neurons

Certain steroids are neuroprotective because they are modulators of neuronal activity or ROS scavengers. We examined neuroprotection following glutamate-induced excitotoxicity in cerebellar granule neuron cultures. 17β-Estradiol, 17α-estradiol (nonestrogenic), or vitamins C+E were equally neuroprotective when coadministered with glutamate, consistent with protection by ROS scavenging. Progesterone protected mainly by an action on GABA-A receptors. Since exogenous antioxidants may influence the level of glutathione, the main endogenous antioxidant in neurons, we investigated if a preconditioning period with the neuroprotectors changed their efficacy as protectors. The neuroprotection by 17β-estradiol and 17α-estradiol, but not progesterone or vitamins C+E, was almost abolished following a preconditioning period of 24 h. This reduction was accompanied by an inhibition of the γ-glutamylcysteine synthetase promoter, and a reduced level of glutathione when preconditioning was combined with the subsequent glutamate exposure. Thus, vitamins C+E and progesterone were more effective long-term neuroprotectors, since preconditioning did not reduce glutathione.     

Peptide deformylase is a potential target for anti-Helicobacter pylori drugs: reverse docking, enzymatic assay, and X-ray crystallography validation

Colonization of human stomach by the bacterium Helicobacter pylori is a major causative factor for gastrointestinal illnesses and gastric cancer. However, the discovery of anti-H. pylori agents is a difficult task due to lack of mature protein targets. Therefore, identifying new molecular targets for developing new drugs against H. pylori is obviously necessary. In this study, the in-house potential drug target database (PDTD, http://www.dddc.ac.cn/tarfisdock/) was searched by the reverse docking approach using an active natural product (compound 1) discovered by anti-H. pylori screening as a probe. Homology search revealed that, among the 15 candidates discovered by reverse docking, only diaminopimelate decarboxylase (DC) and peptide deformylase (PDF) have homologous proteins in the genome of H. pylori. Enzymatic assay demonstrated compound 1 and its derivative compound 2 are the potent inhibitors against H. pylori PDF (HpPDF) with IC50 values of 10.8 and 1.25 microM, respectively. X-ray crystal structures of HpPDF and the complexes of HpPDF with 1 and 2 were determined for the first time, indicating that these two inhibitors bind well with HpPDF binding pocket. All these results indicate that HpPDF is a potential target for screening new anti-H. pylori agents. In addition, compounds 1 and 2 were predicted to bind to HpPDF with relatively high selectivity, suggesting they can be used as leads for developing new anti-H. pylori agents. The results demonstrated that our strategy, reverse docking in conjunction with bioassay and structural biology, is effective and can be used as a complementary approach of functional genomics and chemical biology in target identification.