Microbiological synthesis of stereoisomeric 7(α/ß)-hydroxytestololactones and 7(α/ß)-hydroxytestolactones.

Microbiological synthesis of 7α- and 7ß-hydroxy derivatives of testololactone and testolactone was developed based on bioconversion of dehydroepiandrosterone (DHEA) by fungus of Isaria fumosorosea VKM F-881 with subsequent modification of the obtained stereoisomers by actinobacteria. The first stage included obtaining of the stereoisomers of 3ß,7(α/ß)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones in the preparative amounts. Then the conversion of 7-hydroxylated D-lactones obtained by selected actinobacteria of Nocardioides simplex VKM Ac-2033D, Saccharopolyspora hirsuta VKM Ac-666, and Streptomyces parvulus MTOC Ac-21v was studied. Under the transformation of 3ß,7α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one and its corresponding 7ß-stereoisomer by N. simplex VKM Ac-2033D and S. hirsuta VKM Ac-666 the 7α- and 7ß-hydroxy-17a-oxa-D-homo-androst-4-ene-3,17-dione (7α- and 7ß-hydroxytestololactone), 7α- and 7ß-hydroxy-17a-oxa-D-homo-androsta-1,4-diene-3,17-dione (7α- and 7ß-hydroxytestolactone) were obtained with molar yields in a range of 60.3-90.9 mol%. The crystalline products of 7α-hydroxytestololactone, 7α-hydroxytestolactone, and their corresponding 7ß-hydroxy stereoisomers were isolated, and their structures were confirmed by mass spectrometry and 1H-NMR spectroscopy analyses. The strain of Str. parvulus MTOC Ac-21v transformed 3ß,7(α/ß)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones into the corresponding 3-keto-4-ene analogs and did not show 3-ketosteroid 1(2)-dehydrogenase activity. The activity of actinobacteria towards steroid D-lactones was hitherto unreported.The results contribute to the knowledge of metabolic versatility of actinobacteria capable of transforming steroid substrates and may be applied in the synthesis of potential aromatase inhibitors.

Depression and its related parameters increased the production of autoantibodies against 16α-hydroxyestrone-albumin complex in systemic lupus erythematosus.

Depression is the common and early symptoms associated with early onset of SLE, 16α-hydroxyestrone (16α-OHE1) levels were found to be significantly higher in serum and urine in patients with SLE. This study was carried out in order to know whether depression and its related parameters in the SLE patients enhanced the production of autoantibodies against 16α-OHE1-albumin (A) complexes. The autoantibodies in the serum of 100 SLE [including 65 depressed SLE (DSLE)] patients and 37 control subjects were detected by using direct binding, inhibition ELISA and quantitative precipitin titration. Autoantibodies from DSLE patients (and also the patients who were taken anti-depressant and with neurological symptoms) showed high binding to 16α-OHE1-A in contrast to SLE (p < 0.05) and control subjects (p < 0.001). Although, SLE sera showed high recognition to 16α-OHE1-A in comparison to A (p < 0.05) or 16α-OHE1 (p < 0.001). The affinity of autoantibodies for 16α-OHE1-A was found to be high for DSLE (1.16 × 10-7 M) and SLE (1.24 × 10-7 M) patients as detected by Langmuir plot. The concentration of 16α-OHE1 (p < 0.05) and inflammatory cytokines (IL-6, p < 0.05 and IL-17, p < 0.001) in the serum of SLE patients was found to be significantly higher than controls. Depression and its related parameters in SLE enhanced the production of autoantibodies against 16α-OHE1-A through the generation of inflammatory conditions. Depression in SLE patients increased the release of pro-inflammatory cytokine (IL-6 and IL-17) that in turn generating more autoantibodies and showed strong recognition to 16α-OHE1-A.

Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays.

4-Hydroxyandrost-4-ene-3,17-dione, also named formestane, is an irreversible aromatase inhibitor and therapeutically used as anti-breast cancer medication in post-menopausal women. Currently, no therapeutical indication led to approval of its 17-hydroxylated analog 4-hydroxytestosterone, an anabolic steroid. However, it is currently investigated in a clinical trial for breast cancer. In context with sports doping, aromatase inhibitors are administered to reduce estrogenic side effects of misused anabolic substances or their metabolites. Therefore, both substances are prohibited in sports by the World Anti-Doping Agency (WADA). Analysis of urinary phase I and phase II metabolites showed similar results for both compounds. In the current investigation, 4-hydroxyandrost-4-ene-3,17-dione, 4-hydroxytestosterone and seven of their described urinary metabolites as well as 2α-hydroxyandrostenedione were tested in the yeast androgen screen and the yeast estrogen screen. Androgenic effects were observed for all tested substances, except for one, which showed anti-androgenic properties. With regard to the yeast estrogen screen, estrogenic effects were observed for only two metabolites at rather high concentrations, while six out of the ten substances tested showed anti-estrogenic properties. In terms of the strong androgenic effect observed for 4-hydroxytestosterone (10-8 M), 4-hydroxyandrost-4-ene-3,17-dione (10-8 M) and two more urinary metabolites, the yeast androgen assay may also be used to trace abuse in urine samples.

The identification of 14 alpha,17 beta-dihydroxyandrost-4-en-3-one monohydrate and 14 alpha,17 beta-dihydroxyandrosta-1,4-dien-3-one monohydrate, metabolites of androstenedione in Mucor piriformis.

The microorganism Mucor piriformis transforms androst-4-ene-3,17-dione into a major and several minor metabolites. X-ray crystallographic analysis of two of these metabolites was undertaken to determine unambiguously their composition and chirality. Crystals belong to the orthorhombic space-group P2(1)2(1)2(1), with a = 7.199(4) A and a = 6.023(3) A, b = 11.719(3) A and b = 13.455(4) A, c = 20.409(3) A and c = 20.702(4) A for the two title compounds, respectively. The structures have been refined to final R values of 0.060 and 0.040, respectively.

Cryopreserved human hepatocytes as alternative in vitro model for cytochrome p450 induction studies.

Induction of cytochrome P450 (CYP) by drugs is one of major concerns for drug-drug interactions. Thus, the assessment of CYP induction by novel compounds is a vital component in the drug discovery and development processes. Primary human hepatocytes are the preferred in vitro model for predicting CYP induction in vivo. However, their use is hampered by the erratic supply of human tissue and donor-to-donor variability. Although cryopreserved hepatocytes have been recommended for short-term applications in suspension, their use in studies on induction of enzyme activity has been limited because of poor attachment and response to enzyme inducers. In this study, we report culture conditions that allowed the attachment of cryopreserved human hepatocytes and responsiveness to CYP inducers. We evaluated the inducibility of CYP1A1/2 and CYP3A4 enzymes in cryopreserved hepatocytes from three human donors. Cryopreserved human hepatocytes were cultured in serum-free medium for 4 d. They exhibited normal morphology and measurable viability as evaluated by the reduction of tetrazolium salts (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) by cellular dehydrogenases. Treatment with beta-naphthoflavone (10 microM) for 3 d increased ethoxyresorufin-O-deethylase activity (CYP1A1/2) by 6- to 11-fold over untreated cultures and increased CYP1A2 messenger ribonucleic acid (mRNA) expression by three- to eightfold. Similarly, treatment of cryopreserved human hepatocytes with rifampicin (25 microM) for 3 d increased testosterone 6 beta-hydroxylase activity (CYP3A4) by five- to eightfold over untreated cultures and increased CYP3A4 mRNA expression by four- to eightfold. The results suggest that cryopreserved human hepatocytes can be a suitable in vitro model for evaluating xenobiotics as inducers of CYP1A1/2 and CYP3A4 enzymes.

Double site saturation mutagenesis of the human cytochrome P450 2D6 results in regioselective steroid hydroxylation.

The human cytochrome P450 2D6 (CYP2D6) is one of the major human drug metabolizing enzymes and acts preferably on substrates containing a basic nitrogen atom. Testosterone - just as other steroids - is an atypical substrate and only poorly metabolized by CYP2D6. The present study intended to investigate the influence of the two active site residues 216 and 483 on the capability of CYP2D6 to hydroxylate steroids such as for example testosterone. All 400 possible combinatorial mutations at these two positions have been generated and expressed individually in Pichia pastoris. Employing whole-cell biotransformations coupled with HPLC-MS analysis the testosterone hydroxylase activity and regioselectivity of every single CYP2D6 variant was determined. Covering the whole sequence space, CYP2D6 variants with improved activity and so far unknown regio-preference in testosterone hydroxylation were identified. Most intriguingly and in contrast to previous literature reports about mutein F483I, the mutation F483G led to preferred hydroxylation at the 2ß-position, while the slow formation of 6ß-hydroxytestosterone, the main product of wild-type CYP2D6, was further reduced. Two point mutations have already been sufficient to convert CYP2D6 into a steroid hydroxylase with the highest ever reported testosterone hydroxylation rate for this enzyme, which is of the same order of magnitude as for the conversion of the standard substrate bufuralol by wild-type CYP2D6. Furthermore, this study is also an example for efficient human CYP engineering in P. pastoris for biocatalytic applications and to study so far unknown pharmacokinetic effects of individual and combined mutations in these key enzymes of the human drug metabolism.

11ß-Hydroxydihydrotestosterone and 11-ketodihydrotestosterone, novel C19 steroids with androgenic activity: a putative role in castration resistant prostate cancer?

Adrenal C19 steroids, dehydroepiandrostenedione (DHEA(S)) and androstenedione (A4), play a critical role in castration resistant prostate cancer (CRPC) as they are metabolised to dihydrotestosterone (DHT), via testosterone (T), or via the alternate 5α-dione pathway, bypassing T. Adrenal 11OHA4 metabolism in CRPC is, however, unknown. We present a novel pathway for 11OHA4 metabolism in CRPC leading to the production of 11ketoT (11KT) and novel 5α-reduced C19 steroids - 11OH-5α-androstanedione, 11keto-5α-androstanedione, 11OHDHT and 11ketoDHT (11KDHT). The pathway was validated in the androgen-dependent prostate cancer cell line, LNCaP. Androgen receptor (AR) transactivation studies showed that while 11KT and 11OHDHT act as a partial AR agonists, 11KDHT is a full AR agonist exhibiting similar activity to DHT at 1nM. Our data demonstrates that, while 11OHA4 has negligible androgenic activity, its metabolism to 11KT and 11KDHT yields androgenic compounds which may be implicated, together with A4 and DHEA(S), in driving CRPC in the absence of testicular T.

The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine.

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine. In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.

Identification of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, 4-hydroxy-2-oxohexanoic acid, and 2-hydroxyhexa-2,4-dienoic acid and related enzymes involved in testosterone degradation in Comamonas testosteroni TA441.

Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.

Characterization of testosterone 11 beta-hydroxylation catalyzed by human liver microsomal cytochromes P450.

A combination of accelerator mass spectrometry (AMS) and liquid chromatography-tandem mass spectrometry has been used to clarify some new aspects of testosterone metabolism. The main pathway of testosterone oxidative metabolism by human liver microsomes is the formation of 1beta-, 2alpha-/beta-, 6beta-, 15beta-, and 16beta-hydroxytestosterones, mainly catalyzed by cytochromes P450 2C9, 2C19, and 3A4. We now report the first determination that 11beta-hydroxytestosterone (11beta-OHT) can also be formed by human liver microsomal fractions. The structures of five hydroxylated metabolites of testosterone (2beta-, 6beta-, 11beta-, 15beta-, and 16beta-OHT) and the C-17 oxidative metabolite androstenedione were determined by liquid chromatography with UV detection at 240 nm and liquid chromatography-tandem mass spectrometry. Corresponding results were obtained by high-performance liquid chromatography-AMS analysis of incubations of [4-14C]testosterone with human liver microsomes. 6beta-Hydroxylation was always the dominant metabolic pathway, but 2beta-, 15beta-, and 16beta-OHT, and androstenedione were also formed. The previously undetected hydroxytestosterone, 11beta-OHT, was found to be a minor metabolite formed by human liver microsomal enzymes. It was formed more readily by CYP3A4 than by either CYP2C9 or CYP2C19. 11beta-Hydroxylation was inhibited by ketoconazole (IC50 = 30 nM) at concentrations similar to the IC50 (36 nM) for 6beta-hydroxylation Therefore, CYP3A4 could be mainly responsible for testosterone 11beta-hydroxylation in the human liver. These findings identify human hepatic biotransformation of testosterone to 11beta-OHT as a previously unrecognized extra-adrenal metabolic pathway.

Oxidative fragmentation of pregna-14,16-dien-20-ones to 14 beta-hydroxyandrost-15-en-17-ones.

Two methods have been developed for efficient conversion of pregna-14,16-dien-20-ones into 14 beta-hydroxyandrost-15-en-17-ones. One procedure consists of treatment of the ring-D dienone successively with sodium borohydride and singlet oxygen. The reaction is illustrated by the conversion of pregna-14,16-dien-20-one 1 into 14 beta-hydroxyandrost-15-en-17-one 3, via the corresponding allylic alcohol 2. Although this two-step procedure is simple, it provides 3 in relatively low yield, accompanied by a smaller amount of the isomeric 14 alpha-hydroxyandrost-15-en-17-one 6. An alternative one-step conversion is achieved by treatment of dienone 1 with a peroxyacid in the presence of a strong protic acid. This process is illustrated by the two-step conversion of dienone 1 into hydroxy ketone 11 in 51% overall yield (Scheme 5) and by the analogous conversion of dienone 13 into hydroxy ketone 24 in 61% overall yield (Scheme 11).

Validation of higher-throughput high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry assays to conduct cytochrome P450s CYP2D6 and CYP3A4 enzyme inhibition studies in human liver microsomes.

In the early stage of drug discovery, thousands of new chemical entities (NCEs) may be screened before a single drug candidate can be identified for development. In order to accelerate the drug discovery process, we have developed higher-throughput enzyme assays to evaluate the inhibition of cytochrome P450 isoforms 2D6 (CYP2D6) and 3A4 (CYP3A4) in human liver microsomes. The assays are based on high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) techniques. The analysis time for each sample was reduced from approximately 20 minutes for the conventional HPLC assay to 30 seconds for the LC/MS/MS assay. For both LC/MS/MS assays, the linearity (r(2) > 0.99), precision (%CV < 15%) and accuracy (% bias <15%) for both inter- and intraday validations were satisfactory. Since the implementation of the LC/MS/MS assays, our sample throughput has increased by over 40-fold.

Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation.

The proteome contains hundreds of proteins that in theory could be excellent therapeutic targets for the treatment of human diseases. However, many of these proteins are from functional classes that have never been validated as viable candidates for the development of small molecule inhibitors. Thus, to exploit fully the potential of the Human Genome Project to advance human medicine, there is a need to develop generic methods of inhibiting protein activity that do not rely on the target protein's function. We previously demonstrated that a normally stable protein, methionine aminopeptidase-2 or MetAP-2, could be artificially targeted to an Skp1-Cullin-F-box (SCF) ubiquitin ligase complex for ubiquitination and degradation through a chimeric bridging molecule or Protac (proteolysis targeting chimeric molecule). This Protac consisted of an SCF(beta-TRCP)-binding phosphopeptide derived from IkappaBalpha linked to ovalicin, which covalently binds MetAP-2. In this study, we employed this approach to target two different proteins, the estrogen (ER) and androgen (AR) receptors, which have been implicated in the progression of breast and prostate cancer, respectively. We show here that an estradiol-based Protac can enforce the ubiquitination and degradation of the alpha isoform of ER in vitro, and a dihydroxytestosterone-based Protac introduced into cells promotes the rapid disappearance of AR in a proteasome-dependent manner. Future improvements to this technology may yield a general approach to treat a number of human diseases, including cancer.