A scalable platform to discover antimicrobials of ribosomal origin.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a promising source of new antimicrobials in the face of rising antibiotic resistance. Here, we report a scalable platform that combines high-throughput bioinformatics with automated biosynthetic gene cluster refactoring for rapid evaluation of uncharacterized gene clusters. As a proof of concept, 96 RiPP gene clusters that originate from diverse bacterial phyla involving 383 biosynthetic genes are refactored in a high-throughput manner using a biological foundry with a success rate of 86%. Heterologous expression of all successfully refactored gene clusters in Escherichia coli enables the discovery of 30 compounds covering six RiPP classes: lanthipeptides, lasso peptides, graspetides, glycocins, linear azol(in)e-containing peptides, and thioamitides. A subset of the discovered lanthipeptides exhibit antibiotic activity, with one class II lanthipeptide showing low µM activity against Klebsiella pneumoniae, an ESKAPE pathogen. Overall, this work provides a robust platform for rapidly discovering RiPPs.

Microbial transformation of danazol with Cunninghamella blakesleeana and anti-cancer activity of danazol and its transformed products.

Biotransformation of danazol (1) (17ß-hydroxy-17α-pregna-2,4-dien-20-yno-[2,3-d]-isoxazole) with Cunninghamella blakesleeana yielded three new metabolites 2-4 and a known metabolite 5. These metabolites were identified as 14ß,17ß-dihydroxy-2-(hydroxymethyl)-17α-pregn-4-en-20-yn-3-one (2), 1α,17ß-dihydroxy-17α-pregna-2,4-dien-20-yno-[2,3-d]-isoxazole (3), 6ß,17ß-dihydroxy-17α-pregna-2,4-dien-20-yno-[2,3-d]-isoxazole (4), and 17ß-hydroxy-2-(hydroxymethyl)-17α-pregn-1,4-dien-20-yn-3-one (5). Danazol (1) and its derivatives were evaluated against cervical cancer cell line (HeLa). Compound 1 showed a potent cytotoxicity with IC50=0.283±0.013 µM, as compared to doxorubicin (IC50=0.506±0.015 µM), where compound 3 was also found to be significantly active with IC50=13.427±0.819 µM.

Synergistic effects of danazol and mifepristone on the cytotoxicity of UCN-01 in hormone-responsive breast cancer cells.

BACKGROUND: 7-Hydroxystaurosporine (UCN-01) is a novel antitumor agent as well as a potent inhibitor of a variety of protein kinases with a preference to protein kinase C. Because an intimate interaction exists between PKC signaling and the ER signaling pathways, sex steroid agonists/antagonists might modulate the cytotoxicity of UCN-01. MATERIALS AND METHODS: Effects of sex steroid agonists and antagonists on the UCN-01 cytotoxicity were analyzed by MTT assay in MCF-7/WT and MCF-7/ADR, a multiple drug resistance phenotype lacking ER and PR. RESULTS: MCF-7/ADR is 23-fold more resistant to UCN-01 than MCF-7. In MCF-7/WT, danazol and mifepristone enhanced the cytotoxicity of UCN-01, while these agents did not exert any significant effects on it in MCF-7/ADR. CONCLUSION: These results suggest that danazol or mifepristone might enhance the antitumor activity of UCN-01 in hormone-dependent cancer cells by interacting with PR.

Effects of danazol and medroxyprogesterone acetate on estrogen-(estradiol and estriol) specific binding sites in rabbit uterus.

In rabbit uterus, the presence of separate specific binding sites for not only estradiol but also estriol has been proposed. These sites may be correlated with an antiestradiol effect. Therefore, this study was designed to investigate the effect of antiestrogenic agents such as danazol and medroxyprogesterone acetate (MPA), especially on the estriol binding sites. Danazol and MPA in combination with estradiol were administered subcutaneously to immature female rabbits daily for 10 days, and resulted in a significant (p < 0.05) decrease in uterine weight and estradiol binding sites in the uterus. Treatment with MPA significantly (p < 0.05) decreased the level of estriol binding sites, but treatment with danazol resulted in this to a minimal extent in the uterus primed by estradiol. MPA did not bind to estradiol and estriol binding sites, while danazol at a high concentration bound to estriol binding sites with some affinity, but not to estradiol binding sites in the uterine cytosol of estrogen-primed rabbits. These results suggest that within the antiproliferative effect of danazol and MPA (an antiestrogenic action on estrogen-stimulated uterine growth) there are likely to be specific differences between some of the possible mechanisms of danazol and MPA in their action at the estriol binding site.

Adrenal steroids in serum during danazol therapy, taking into account cross-reactions between danazol metabolites and serum androgens.

To investigate the changes in the serum androgen concentrations and the Free Androgen Index (FAI) in women during danazol therapy, we measured the serum concentrations of adrenal steroids and danazol metabolites, and then examined the effects of danazol metabolites on assays for serum androgens. Thirteen women who had endometriosis were treated with danazol (300 or 400 mg/day) for 8 to 16 weeks. Blood samples were taken before, during, and after the medication. During the danazol therapy, serum testosterone (T), cortisol (F), and sex-hormone binding globulin (SHBG) significantly decreased (P < 0.05); but serum dehydroepiandrosterone-sulfate (DHEAS) and FAI increased (P < 0.05). The serum concentrations of danazol metabolites were: danazol, 209.0 +/- 28.3 (ng/mL, mean +/- SEM); delta 1-2-hydroxymethyl ethisterone, 114.4 +/- 8.4; and 2-hydroxymethyl ethisterone, 660.0 +/- 54.2. There was considerable cross-reaction between danazol metabolites and androgens [T, androstenedione (A), and dehydroepiandrosterone (DHEA)] in the direct assays. As for the ratios of adrenal steroids in serum, the DHEAS/F, DHEAS/DHEA, and 11-deoxycortisol (S)/F ratios increased (P < 0.05). We conclude that the increase in FAI and DHEAS represents increased native androgenic activity with danazol, and the changes in adrenal steroid ratios in serum indicate the inhibition of 11 beta-hydroxylase and sulfatase activities during danazol therapy.

[Immunohistochemical determination of endometrial progesterone receptor (PR) content after intrauterine infusion of danazol in rabbits].

Danazol, an isoxazole derivative of 17 alpha-ethinyl-testosterone, has various effects on the female reproductive system. To examine its anti-estrogenic effects through the receptor system and clarify its direct effect on the endometrium in vivo, we applied danazol jelly into the rabbit uterine cavity. Recent studies performed in our laboratory using the ligand-binding assay have shown that danazol administered directly into the uterine cavity is absorbed by the endometrial tissue and exerts its anti-estrogenic effects possibly through progesterone receptors (PR) in the cells. In the present study we examined the variations in endometrial PR content during 7 days after the intrauterine administration of danazol into the rabbit uterine cavity. Immature female white rabbits were used. They were given daily subcutaneous injections of estradiol-17 beta (E2) for 3 days after which their abdomens were opened, and danazol gel was infused into the lumen of the right horn and HPC-jelly was infused into the lumen of the left horn, as the control. Thereafter and until they were killed, the same daily dose of E2 was injected. They were killed 6h, 24h, 3 days, 5 days or 7 days after intrauterine infusion of danazol and PRs were studied immunohistochemically using a specific antireceptor monoclonal antibody (PR-AT 4.14). Six hours after danazol infusion, the PR staining in the glandular epithelium and part of the stroma surrounding the epithelium had declined. On day 3, PR staining declined to its lowest level in the glandular epithelium and stroma. On Day 5, PR staining had been restored in the glandular epithelium but stayed low in the stroma.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between oestradiol and danazol on the growth of gastrointestinal tumour cells.

The gastrointestinal tumour cell lines, MKN45G and C146 possessed oestrogen receptors (ER) of affinities 1.8 x 10(-8) and 5.2 x 10(-9) M respectively. C146 and MKN45G had enhanced proliferation in the presence of oestradiol (10(-8) and 10(-10) M). Fifty-six percent (5/9) of primary gastric and colorectal tumours had their proliferation enhanced by oestradiol. The anti-steroidal drug, danazol, inhibited the basal growth of MKN45G (10 micrograms ml-1 45% of the untreated control), C146 (10 and 5 micrograms ml-1, 20 and 48% of control, respectively) and 3/9 GI primary tumours (10 micrograms ml-1). Danazol competed with 3 [H]-Oestradiol for binding to ER on MKN45G and C146, at concentrations from 10 to 1 micrograms ml-1.

Pseudohypertestosteronemia during danazol therapy. A case report.

Several steroid radioimmunoassay kits, particularly testosterone, cross-react with danazol, producing false elevations of serum steroid levels. A patient taking danazol simultaneously developed signs of virilization and an apparent 15-fold elevation in her serum testosterone. Discontinuation of danazol resulted in a prompt return to physical and biochemical normality.

Comparison of the pharmacology of nafarelin and danazol.

The pharmacologic profiles of danazol and nafarelin differ considerably from each other. Danazol interacts with multiple classes of proteins, whereas the gonadotropin-releasing hormone agonist nafarelin interacts only with the pituitary gonadotropin-releasing hormone receptor. Differences in the molecular, endocrine, and clinical pharmacologic properties of these agents may provide clues to their varying effects in the management of women with endometriosis.

Growth inhibition by danazol in a human endometrial cancer cell line with estrogen-independent progesterone receptors.

Since we recently found that danazol, an isoxazol derivative of ethinyltestosterone, has a growth-inhibitory effect on human endometrial cancer cells in primary culture, the effects of danazol on a human endometrial cancer cell line (IK-90 cells), which contains estrogen-independent progesterone receptors (PR), were investigated in the present study. The addition of danazol (1 nM-1 microM) in culture medium caused a decrease in the growth of IK-90 cells in a dose-dependent manner. Competitive binding studies showed that danazol effectively binds to PR in IK-90 cells, and the binding affinity for PR was estimated to be 6.0% of that of R5020. The addition of 1 microM danazol in culture medium resulted in a rapid and significant increase in nuclear PR with a concomitant decrease in cytoplasmic PR in the cells. These findings suggest that danazol has a growth-inhibitory effect on human endometrial adenocarcinoma cells directly through PR system in the cells.

Modelo experimental de endometriose produzido por técnica microcirúrgica em ratas albinas: estudo anatomopatológico dos implantes sob açäo de danazol, valerianato de estradiol e ooforectomia / Experimental model of endometriosis produced by microsurgery in albino rats: anatomopathologic study of the implants under the effect of danazol, estradiol valeriato and oophorectomy

Com o objetivo de estabelecer um modelo experimental de endometriose cirúrgica e de se estudar a açäo do danazol, valerianato de estradiol e ooforectomia, foram empregadas 48 ratas albinas submetidas a implante endometrial cirúrgico no peritônio lateral esquerdo, distribuídos em 04 grupos experimentais: controle, ooforectomia, danazol e valerianato de estradiol. Utilizou-se a citologia vaginal como parâmetro para indicaçäo do ato cirúrgico. O fragmento de endométrio foi obtido do corno direito e implantado no peritônio lateral esquerdo por meio de microcirurgia. O sacrifício dos animais foi realizado 30 dias após a cirurgia e realizado necroscópia de todos os órgäos. O material do implante foi estudado histopatologicamente. Durante o experimento analisou-se os seguintes parâmetros clínicos: citologia vaginal, glândulas mamárias e secreçäo vaginal. A análise dos resultados obtidos demonstrou exequibilidade do modelo experimental permitindo deduzir que o estímulo com o valerianato de estradiol mostre presença de tecido glandular com elevado índice de mitose, alteraçäo como hiperplasia do implante endometrial cirúrgico. Sob a influência do danazol os implantes apresentaram-se inviáveis, mostrando fibrose intensa

Danazol binds to progesterone receptors and inhibits the growth of human endometrial cancer cells in vitro.

Based on our recent findings that danazol, an isoxazol derivative of ethinyltestosterone, has a profound growth-inhibitory effect on an established human endometrial adenocarcinoma cell line, the effects of danazol on cancer cells from human endometrial adenocarcinomas obtained by hysterectomy were investigated in the present study. Of the 22 uterine adenocarcinomas, estrogen, progesterone, and androgen receptors were found in 12, 14, and 4 tumors, respectively. Competitive binding studies showed that danazol specifically binds to progesterone and androgen receptors but not to estrogen receptors. Of the five cancer cells from five patients succeeded in primary cell culture, a marked inhibition of cell growth was demonstrated by addition of danazol in two cancer cells having progesterone but not androgen receptors. However, danazol did not affect the growth of the remaining three cancer cells lacking progesterone receptors. These results strongly suggest that danazol has a significant growth-inhibitory effect on human endometrial adenocarcinoma cells, possibly through progesterone receptors in the cells.

[Interaction of blood sex steroid-binding globulin-steroid complexes with plasma membranes of human decidual endometrium cells]. / Vzaimodeistvie kompleksov sekssteroidsviazyvaiushchii globulin krovi-steroid s plazmaticheskimi membranami kletok detsidual'nogo éndometriia cheloveka.

Plasma membranes of decidual tissue cells specifically bind the sex steroid-binding globulin (SBP) complexes with estrogen (estradiol, estriol, estrone) and with the pharmacological agent danazol but do not interact with the SBP-testosterone or SBP-dihydrotestosterone complexes. The selectivity of interaction of the SBP-steroid complexes with decidual tissue cellular membranes provide evidence for the active role of SBP in the realization of steroid effects on the target tissue.

Effects of progesterone, progestogens, and danazol on the specific cortisol binding in human plasma.

The interaction of medroxyprogesterone acetate (MPA) with cortisol binding to corticosteroid-binding globulin (CBG) was studied with the use of an aqueous two-phase system with polyethylene glycol and dextran for equilibrium partition. Competitive binding analyses were also performed for progesterone (P), levonorgestrel, norethisterone, danazol, and tamoxifen. P and danazol were found to exert cortisol displacing activity, whereas MPA and the other tested compounds had no such effect. The glucocorticoid effects reported for MPA could not be explained by displacement. In general, P serum concentrations are lower than those of cortisol, and most binding sites on CBG are occupied by the glucocorticoid. At high P levels displacement and an increase in free cortisol may occur. Danazol displacement of cortisol is hampered by its pronounced albumin binding. In conclusion, none of the tested compounds should increase free and biologically active cortisol during normal clinical treatment.

[Changes in % free testosterone and sex-hormone binding globulin during danazol administration].

To clarify the androgen balance on the administration of danazol a study was conducted on the levels of % free testosterone (% free T) and sex-hormone-binding globulin (SHBG) during the therapy and the binding properties of danazol to SHBG and androgen receptor. It was found that danazol displaced testosterone (T) from SHBG and R1881 from androgen receptor, in vitro. During treatment with danazol, levels of % free T were increased about three fold, but in contrast the SHBG concentration were significantly decreased. Similar changes in % free T were observed by computer simulation. These data suggest that the androgenic effects of danazol were the results of increased free T levels by the T-displacing ability of danazol from SHBG. The concentration of SHBG might be reduced as a consequence of increased levels of free T. On the other hand, danazol binding to androgen receptor might partially inhibit the action of excess amounts of free T, because the androgenization during therapy was too weak to compare the levels of free T.

Danazol binding to steroid receptors in human uterine endometrium.

To understand the mechanism of action of danazol, the binding of danazol to multiple classes of intracellular steroid binding proteins was studied in the human uterine endometrium. Danazol bound to endometrial receptors for estrogen, progesterone, and androgen and seemed to bind to endometrial intracellular corticosteroid-binding globulin and sex-hormone-binding globulin. Danazol occupies almost all binding sites of steroids in the steroid target cells in spite of the presence of endogenous steroids. It is speculated that the binding behavior of danazol may be related to its therapeutic effect on endometriosis.

PIP: Binding of danazol to multiple classes of intracellular steroid binding proteins was studied in the human uterine endometrium in an effort to understand danazol's mechanism of action. Danazol, which binds to human endometrial cytosol receptors for androgen (AR), progestin (PR), and estrogen (ER), also appears to interact with sex hormone binding globulin and corticosteroid binding globulin present in the cytosol preparations. 17 beta-hydroxy-17 alpha-methyl-estra-4, 9, 11-trine-3-one-[17 alpha-methyl-3h], 87 Ci/mmol (3H-R1881) binds to AR and PR in the human uterine endometirum. Thus, 3H-R1881/PR binding was abolished by the presence of progesterone. Previously reported studies have demonstrated that danazol binds with relatively high affinity to AR. Also, the danazol/AR complex can translocate to the nucleus. These findings are consistent with the observed androgenic effects of danazol in rats and women. Danazol binds with moderate affinity to PR. The danazol/PR complex is poorly translocated to the nucleus, suggesting that danazol is antiprogestational. Yet, a major metabolite of danazol, ethisterone, is progestational. The progestational vs. the antiprogestational effects of danazol remain controversial. Danazol displacement of 6, 7-3H-)stradial-17 beta (3H-E2) from ER is not parallel to E2 displacement of 3H-E2 when compared with that of PR or AR. This discrepancy may derive from an affinity difference. Danazol binds poorly to ER, yet circulating concentrations of danazol in vivo may lead to complete occupancy of the ER by danazol, which may interfere with normal endometrial ER dynamics, resulting in an antiestrogenic effect. Danazol occupies almost all binding sites of steroids in the steroid target cells despite the presence of endogenous steroids. The binding behavior of danazol may be related to its therapeutic effect on endometriosis.

The interaction of human endometrial and myometrial steroid receptors with danazol.

The affinity of danazol for oestrogen, androgen and progesterone receptors in human endometrium and myometrium was determined, to study the mechanism of action of this drug in the treatment of endometriosis. The ability of danazol to combine with each of the three types of receptor was similar in both endometrium and myometrium. The capacity of danazol to compete with oestradiol-17 beta for the oestrogen receptor was very low (1.72 +/- 0.48 X 10(-3%) cross reaction, mean +/- SEM) and danazol, at the maximum concentration used, was unable to saturate the receptor; but danazol's ability to compete with progesterone for its receptor was considerably higher (8.41 +/- 1.65% using progesterone, 1.95 +/- 0.41% using R5020) and was saturable. Danazol was also able to displace dihydrotestosterone from the cytosol androgen receptor (6.29 +/- 1.82% cross reaction). The association constant of oestradiol for the endometrial and myometrial oestrogen receptors was 2.19 X 10(9)M-1 and 7.45 X 10(9)M-1 respectively, while that of progesterone and dihydrotestosterone for their receptors was similar in endometrium and myometrium (mean 0.25 +/- 0.06 X 10(9) M-1 and 3.62 +/- 1.67 X 10(9) M-1 respectively). Using R5020, the association constant for the myometrial progesterone receptor was 2.50 +/- 0.73 X 10(9) M-1. We conclude that, in view of the high circulating levels of danazol present in patients being treated for endometriosis, it is possible that danazol may bind to, and partly saturate, endometrial and myometrial oestrogen, progesterone and androgen receptors during treatment. An explanation may thus be provided for some of the diverse actions of this drug.

Further characterization of estrogen receptors in the human oviduct.

Estrogen binding in human oviducts was studied in vitro by the dextran-coated charcoal assay and sucrose density ultracentrifugation. Estrogen binds with high affinity and limited capacity to cytosol of the human oviduct. The concentration of competitive inhibitors to produce 50% reduction in estrogen binding was 8 x 10(-8) M for the antiestrogen CI-628, 8 x 10(-7) M for the progestogen norethynodrel, and 3 x 10(-6) M for the testosterone derivative danazol at the ligand concentration of 1 nM estradiol. Nuclear estrogen binding was not inhibited by a 100-fold excess of progesterone or by a 10-fold excess of norethynodrel. Estrogen-binding protein with a sedimentation coefficient of 4S was seen in oviductal cytosol of all three anatomic segments. The nuclear 4S peak of estrogen binding was demonstrated in the ampullary tubal segment.

Free testosterone levels during danazol therapy.

Danazol is a testosterone (T) derivative widely used in the clinical treatment of endometriosis. Its mechanism of action is poorly understood, but is side effects are mainly androgenic. Previously it was demonstrated that danazol can displace T from sex-hormone-binding globulin (SHBG). The binding properties of danazol to SHBG and albumin were studied with the use of labeled danazol in an aqueous two-phase equilibrium partition system. Levels of total T, SHBG, and albumin were measured in 16 women undergoing danazol treatment for endometriosis. Thereafter, free and protein-bound T levels were calculated. A marked rise in free T was found during danazol therapy as compared with pretreatment levels. The data suggest that many of the effects of danazol could be explained by increased levels of free T during treatment.