Antibacterial and antibiofilm efficacy of repurposing drug hexestrol against methicillin-resistant Staphylococcus aureus.

There has been an explosion in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) because of the indiscriminate use of antibiotics. In this study, we repurposed hexestrol (HXS) as an antibacterial agent to fight planktonic and biofilm-related MRSA infections. HXS is a nonsteroidal synthetic estrogen that targets estrogen receptors (ERα and ERß) and has been used as a hormonal antineoplastic agent. In our work, the minimum inhibitory concentrations (MICs) were determined using the antimicrobial susceptibility of MSSA and MRSA strains. Anti-biofilm activity was evaluated using biofilm inhibition and eradication assays. Biofilm-related genes were analyzed with or without HXS treatment using RTqPCR analysis of S. aureus. HXS was tested using the checkerboard dilution assay to identify antibiotics that may have synergistic effects. Measurement of ATP and detection of ATPase allowed the determination of bacterial energy metabolism. As shown in the results, HXS showed effective antimicrobial activity against S. aureus, including both type strains and clinical isolations, with MICs of 16 µg/mL. Sub-HXS strongly inhibited the adhesion of S. aureus. The content of extracellular polymeric substances (EPS) and the relative transcription levels of eno, sacC, clfA, pls and fnbpB were reduced after HXS treatment. HXS showed antibacterial effects against S. aureus and synergistic activity with aminoglycosides by directly interfering with cellular energy metabolism. HXS inhibits adhesion and biofilm formation and eradicates biofilms formed by MRSA by reducing the expression of related genes. Furthermore, HXS increases the susceptibility of aminoglycosides against MRSA. In conclusion, HXS is a repurposed drug that may be a promising therapeutic option for MRSA infection.

A yeast-based screening assay identifies repurposed drugs that suppress mitochondrial fusion and mtDNA maintenance defects.

Mitochondria continually move, fuse and divide, and these dynamics are essential for the proper function of the organelles. Indeed, the dynamic balance of fusion and fission of mitochondria determines their morphology and allows their immediate adaptation to energetic needs as well as preserving their integrity. As a consequence, mitochondrial fusion and fission dynamics and the proteins that control these processes, which are conserved from yeast to human, are essential, and their disturbances are associated with severe human disorders, including neurodegenerative diseases. For example, mutations in OPA1, which encodes a conserved factor essential for mitochondrial fusion, lead to optic atrophy 1, a neurodegeneration that affects the optic nerve, eventually leading to blindness. Here, by screening a collection of ∼1600 repurposed drugs on a fission yeast model, we identified five compounds able to efficiently prevent the lethality associated with the loss of Msp1p, the fission yeast ortholog of OPA1. One compound, hexestrol, was able to rescue both the mitochondrial fragmentation and mitochondrial DNA (mtDNA) depletion induced by the loss of Msp1p, whereas the second, clomifene, only suppressed the mtDNA defect. Yeast has already been successfully used to identify candidate drugs to treat inherited mitochondrial diseases; this work may therefore provide useful leads for the treatment of optic atrophies such as optic atrophy 1 or Leber hereditary optic neuropathy.

The influence of hexestrol diacetate on gametogene function in male rabbit.

The research was performed on 24 male sexually mature domestic rabbits, divided in two equal batches. The rabbits from the first batch were administered 0.5 mg/kg of body weight hexestrol diacetate intramuscularly twice a week for four consecutive weeks. Batch 2 was used for control. The testicular tissue samples obtained after orchidectomy were processed in order to obtain histological samples, stained using the Goldner's trichrome method. Examination of histological sections from the control batch showed that a natural aspect of seminal tubule epithelium, without reported injuries, even discrete. In the experimental batch were recorded a number of changes to all categories of the seminal cells. The severity and extent of damages varied greatly from one seminal tube to another and even from one part to another of the same seminal tube. These changes were the appearance of apoptotic cells and apoptotic bodies, vacuolar degenerescence of spermatocytes and spermatides, syncitialisation of spermatides, areas of necrosis accompanied by severe disruption of the seminiferous epithelium. In some areas, the lesions were so severe that the affected area of the cells forms "basal area". If lesions in the "adluminal area" affects only temporarily gametogenetic function the lesions in the "basal area" are irreversible as there are named "reserve cells" which is the starting point of spermatogenesis. Highlighted issue raised on the opportunity use of hexestrol diacetate in therapy or animal production stimulation as it gametogenetic function in males while their risk transfer to humans through consumption of foods of animal origin.

[Effects of Yi Fu Ning soft gelatin capsules on reproductive endocrine-immune function of ovariectomized rats].

OBJECTIVE: To investigate the effect of Yi Fu Ning Soft Gelatin Capsules (YFN) on reproductive endocrine-immune function of ovariectomized rats. METHODS: 60 3-month old female Sprague-Dawley rats were used, 50 of them were ovariectomized and randomly divided into 5 groups: ovariectomizy (OVX) group, OVX with diethylstilbestrol tablets (DT) group, OVX with YFN (high dose, middle dose and low dose) group. The others were sham-operated group. The rats were administrated initially in the 4th week after the operation. After drugs had been given for 12 weeks the rats were sacrificed, blood serum hormone, IL-2 content and T lymphocyte subpopulation were detected with methods radioimmunoassay and flow cytometry (FCM). RESULTS: (1) Compared with sham group, the level of serum E2, Te and P significantly decreased (P < 0.01), FSH, LH content significantly increased; Blood T lymphocyte subpopulation CD3+ cells, CD4+ cells and CD4+/CD8+ ratio significantly decreased, serum IL-2 content also significantly decreased (P < 0.01). (2) Compared with model group, after treated by YFN, the level of serum E2 and P significantly increased (P < 0.01), serum FSH and LH content significantly decreased; T lymphocyte subpopulation CD3+ cells, CD4+ cells and CD4+/CD8+ ratio were improved significantly and serum interleukin-2 (IL-2) content increased significantly. CONCLUSION: YFN can increase serum sexual hormone content,reduce the level of FSH and LH, and improve imbalanced T lymphocyte subpopulation, stimulate IL-2 excretion, which means YFN can regulate inordinate reproductive endocrine-immune network in ovariectomized rats.

Structure of the G225P/G226P mutant of mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) ternary complex: implications for the binding of inhibitor and substrate.

3(17)alpha-Hydroxysteroid dehydrogenase (AKR1C21) is a unique member of the aldo-keto reductase (AKR) superfamily owing to its ability to reduce 17-ketosteroids to 17alpha-hydroxysteroids, as opposed to other members of the AKR family, which can only produce 17beta-hydroxysteroids. In this paper, the crystal structure of a double mutant (G225P/G226P) of AKR1C21 in complex with the coenzyme NADP(+) and the inhibitor hexoestrol refined at 2.1 A resolution is presented. Kinetic analysis and molecular-modelling studies of 17alpha- and 17beta-hydroxysteroid substrates in the active site of AKR1C21 suggested that Gly225 and Gly226 play an important role in determining the substrate stereospecificity of the enzyme. Additionally, the G225P/G226P mutation of the enzyme reduced the affinity (K(m)) for both 3alpha- and 17alpha-hydroxysteroid substrates by up to 160-fold, indicating that these residues are critical for the binding of substrates.

Effects of hexestrol on mouse ovarian morphology and ovulation.

OBJECTIVE: To analyze histological aspects of ovaries as well as the ovulation of adult mice treated with the anabolic agent hexestrol. METHODS: Thirty adult mice were divided into three groups of 10 animals each: (GI) the animals received a dose of 3 mg/kg of hexestrol; (GII) the animals were given a dose of 6 mg/kg of hexestrol; (GIII) the animals were injected with distilled water (vehicle). Another 10-animal group (GIV) was included, and these mice were injected with propionate testosterone (1.25 mg) after 5 days from the day of birth. Hexestrol was administered intraperitoneally once a day and the treatment lasted 30 days. The mice were then sacrificed; their ovaries and oviducts were removed, submitted to histological routine and analyzed under light microscopy. RESULTS: In mice treated with hexestrol (6 mg/kg) (Group II), ovaries were smaller than those from the controls but highly vascularized; similar results were obtained in GIV. A great number of follicles in several stages of development were found -- however, with no corpora lutea -- in six animals in GII. No corpora lutea were found in GIV. The number of luteal bodies and oocytes in GII was lower than that in GI or GIII. No oocytes were detected in GIV. Finally, the nuclear volume of interstitial cells in GII and GIV was the largest. CONCLUSION: Our data suggest that the anabolic agent hexestrol in a high dose may decrease ovulation in mice.

[Hexestrol inhibits ATP-induced contractile response of isolated preparations of pregnant human uterus].

The results of in vitro experiments showed that hexestrol, a synthetic analog of estrogen hormones, at high concentrations inhibited the contractile response of isolated preparations of pregnant human uterus induced by ATP. It is suggested that estrogen hormones can interact with P2 receptors in human uterus during pregnancy.

[Inhibitory effect of zinc on beta-hexosaminidase release from RBL-2H3 cells by synthetic chemicals].

The effects of foods and chemicals related to food hygiene on degranulation were evaluated using a method for assaying the enzyme activity of beta-hexosaminidase as an index of chemical mediator release from RBL-2H3 cells in vitro. Using a previously developed assay system, we had found a large number of inhibitors and promoters of degranulation of RBL-2H3 cells. In the present study, we examined the inhibitory effect of zinc chloride on the degranulation (beta-hexosaminidase release) from RBL-2H3 cells with or without antigen in the presence of the degranulation-promotive chemicals, namely, 4 food additives, 7 pesticides and 2 veterinary drugs. These promotive chemicals were classified into two types on the basis of inhibitory profile by zinc chloride: 1) those which showed marked degranulation-inhibitory action when the cells were stimulated with antigen, such as butylhydroxyanisole, dibutylhydroxytoluene, EPN, cis- and trans-permethrin, prothiofos, pyridaben, terbufos, 2) those which showed marked degranulation-inhibitory action whether the cells were stimulated with antigen or not, such as butyl p-hydroxybenzoate, o-phenylphenol, bitertanol, salinomycin. In conclusion, zinc had a dramatic inhibitory effect on enhanced degranulation induced by synthetic chemicals in vitro.

[Effects of veterinary drugs on beta-hexosaminidase release from rat basophilic leukemia cells (RBL-2H3)].

Little is known about the effects of residual veterinary drugs on the allergic reaction, except for the antigenicity of antibiotics and synthetic antimicrobials. Therefore, 59 kinds of veterinary drugs were investigated for their effects on the IgE receptor-mediated beta-hexosaminidase release from RBL-2H3 cells as an index of immediate allergic reaction. We found that the antibiotics chlorotetracycline, doxycycline, monensin, the synthetic antimicrobial pyrimethamine and the steroid hormone testosterone inhibited beta-hexosaminidase release. Most of the veterinary drugs showed no action, though the ionophores lasalocid, salinomycin and the steroid hormone hexestrol promoted beta-hexosaminidase release from injured cells. Based on the residual levels of these drugs and the frequencies of detection in actual food samples, it seems unlikely that these drugs have any immediate allergic effect in practice.

Positive and negative discrimination of estrogen receptor agonists and antagonists using site-specific DNA recombinase fusion proteins.

Activation of the estrogen receptor (ER) by hormone involves at least two steps. First, hormone binding initially relieves repression, a property imposed on ER in cis by its ligand-binding domain (EBD). Subsequently, the derepressed ER binds specific genomic sites and regulates transcription. In addition to the natural hormone, ER binds a broad range of ligands that evoke a spectrum of responses ranging from full ER activation by agonists to partial activation and inhibition by partial or complete antagonists. How these different ligands evoke different ER responses remains unclear. To address this issue, we have developed a nontranscriptional assay for ER ligand responsiveness based on Flp recombinase/human EBD protein chimeras. These fusion proteins transduce the transient event of ligand binding into a permanent DNA change in a human cell line system. A fusion protein including ER D, E, and F domains was activated by all the ER ligands tested, demonstrating that both agonists and antagonists serve to relieve initial repression, and that differences between them lie downstream in the activation pathway. Mutant variants of the Flp-ER protein that distinguish between agonists and antagonists, and a mutant EBD that selectively lost the ability to respond to 17beta,-estradiol but not to other ligands, were also identified. Thus, agonists and antagonists can be functionally distinguished in a nontranscriptional assay.

Endocrine mechanisms of suppressive effect of low dose estrogen-antiandrogen treatment on androgen-dependent organs of male rats.

The effects of low doses of hexestrol (Hex) (2-40 micrograms/kg bw) and flutamide (FI) (10 mg/kg bw) on some endocrine mechanisms in mature intact male rats are described in the present paper. It has been shown that each preparation, administered separately for 10 days, induced a moderate decrease in the weight of the ventral prostate (VP), anterior prostate lobe or coagulating gland (CG) and seminal vesicles (SV), in the DNA content and number of cells in the VP. 5 alpha-reductase activity was also decreased; the epithelium secretory activity of the VP was suppressed. After combined FI (10 mg/kg bw) and Hex (40 micrograms/kg bw) the following castration-like effects were observed: an abrupt fall in the weight of the accessory sexual glands, a decrease of DNA level and cell number in the VP as well as a suppression of the production of 5 alpha-reductase metabolites in this structure. Histologically, a marked degenerative changes in the VP secretory epithelium was observed; on the contrary an hyperplasia of connective and smooth muscle cells was evident. When FI alone was administered to rat, the above-mentioned changes were accompanied by a pronounced elevation of plasma bio-LH and testosterone (T) levels, also an increase of testicular delta 5-3 beta-hydroxysteroid dehydrogenase activity was observed. On the contrary, when Hex was administered alone or in combination with FI, bio-LH and T levels and enzyme activity decreased. We conclude that Hex administration in low doses, in combination with FI, could be an alternative method for a complete androgen blockade of the accessory sexual glands.

Aminoglycoside antibiotics prevent the formation of non-bilayer structures in negatively-charged membranes. Comparative studies using fusogenic (bis(beta-diethylaminoethylether)hexestrol) and aggregating (spermine) agents.

Aminoglycoside antibiotics cause aggregation but not fusion of negatively-charged liposomes at an extent proportional to their capacity to interact with acidic phospholipids (Van Bambeke et al., 1995, Eur. J. Pharmacol., 289, 321-333). To understand why aggregation is not followed by fusion, we have examined here the influence of two aminoglycosides with markedly different toxic potential (gentamicin > isepamicin) on lipid phase transition in negatively-charged liposomes using 31P-NMR spectroscopy, in comparison with spermine (an aggregating agent) and bis(beta-diethylaminoethylether)hexestrol or DEH (a fusogenic cationic amphiphile). Gentamicin, spermine, and, to a lesser extent, isepamicin inhibit the appearance of the isotropic signal seen upon warming of control liposomes and denoting the presence of mobile structures. This non-bilayer signal appeared most prominently when liposomes were incubated with DEH, a strong fusogenic agent. We conclude that aminoglycosides, like spermine, have the potential to prevent membrane fusion, by inhibiting the development of a critical change in membrane organization, which is associated with fusion. We suggest that this capacity could be a determinant in aminoglycoside toxicity.

Carcinogenic antioxidants. Diethylstilboestrol, hexoestrol and 17 alpha-ethynyloestradiol.

The synthetic oestrogens diethylstilboestrol, hexoestrol and 17 alpha-ethynyloestradiol are known to be carcinogenic, yet they all exert antioxidant properties in vitro in that they are good inhibitors of iron ion-dependent lipid peroxidation. In rat liver microsomes incubated with Fe(III)-ascorbate or Fe(III)-ADP/NADPH and also in ox-brain phospholipid liposomes incubated with Fe(III)-ascorbate; the overall order of effectiveness of the compounds tested as inhibitors of lipid peroxidation was diethylstilboestrol > hexoestrol > 17 alpha-ethynyloestradiol > 4-hydroxytamoxifen > 17 beta-oestradiol > tamoxifen. Compounds acting as antioxidants towards lipids may also exert pro-oxidant effects towards other molecules such as DNA and thus must never be assumed to be safe for human use.

Structure-activity relationship of 3,3'-dihydroxy-alpha,beta-diethyldiphenylethane and its related compounds.

3,3'-Dihydroxy-alpha,beta-diethyldiphenylethane (I), 3,3'-dihydroxy-alpha,beta-diethylstilbene (II), hexestrol (III) and diethylstilbestrol (IV) have already been reported to show hypotensive effects on rats and exhibit phytogrowth-inhibitory activities. We have proposed that two phenolic hydroxyl groups in these compounds are necessary for the biological activities, and a structure-activity relationship for I-related compounds was accomplished using molecular-mechanics (MM) calculations. As a result, the following three findings were obtained; 1) the minimized conformational energy obtained from MM calculations, which is a parameter expressing the molecular stability, showed a relatively high correlation with the biological activities, 2) as results of quantitative structure-activity relationship (QSAR) analyses, the combination of the distance between two phenolic hydroxyl oxygens led to the regression equations with high correlation values, and 3) the idealized molecular model of the most active compound (I) showed the highest stability and had a particular conformation which differed from the other compounds (II-IV).

Effect of diethylstilbestrol and related compounds on the Ca(2+)-transporting ATPase of sarcoplasmic reticulum.

Diethylstilbestrol is a potent inhibitory agent of the Ca(2+)-ATPase activity of sarcoplasmic reticulum membranes. Other structurally related molecules, such as dienestrol or hexestrol having hydroxyl groups at para positions of the two benzene rings produce similar effects. The absence or derivatization of the hydroxyl groups as occurs with trans-stilbene or diethylstilbestrol dipropionate converts the structure in an activating agent of the enzyme. The Ca2+ transport profiles in the presence of the referred drugs reproduces the same behavior observed for the hydrolytic activity. There is also a clear indication of a membrane-mediated mechanism of these drugs. Ligand binding experiments at equilibrium indicate that diethylstilbestrol decreases the affinity for Ca2+ of the high affinity Ca2+ sites. Functional studies reveal that the activation/inhibition induced by these drugs is correlated with decreased levels of phosphoenzyme at steady state, and these levels are sensitive to the Ca2+ concentration. Chase experiments of [32P]phosphoenzyme and 45Ca2+ indicate a slight activation effect of diethylstilbestrol dipropionate on Ca2+ dissociation during the enzyme turnover. The use of different anthroyloxy derivatives of stearic acid as a fluorescent probe suggest that diethylstilbestrol and other inhibitory agents could be located close to the polar region of the lipid bilayer, which interferes with the Ca(2+)-binding sites, whereas the activators trans-stilbene and diethylstilbestrol dipropionate may have a deeper position into the membrane, which accelerates the Ca2+ translocation process.

Qualitative study of the interaction mechanism of estrogenic drugs with tubulin.

Synthetic estrogenic drugs (E-diethylstilbestrol, erythro-hexestrol and E,E-dienestrol) inhibit tubulin assembly and erythro-hexestrol and E,E-dienestrol lead to the formation of twisted ribbon structures. For the inhibitory effect on tubulin assembly, estrogenic drugs seem to interact directly with tubulin 6S on site(s) analogous to the colchicine-site, but independent of the GTP- and vinblastine-sites. This binding does not involve tubulin tryptophanyl residues or sulfhydryl groups. The influence of temperature, calcium and magnesium on the formation of twisted ribbon structures induced by the binding of estrogenic drugs to microtubular protein and tubulin has also been studied. This formation is strongly magnesium-dependent whereas preformed twisted ribbon structures are calcium- and chilling-insensitive.

Uterine adenocarcinoma in mice following developmental treatment with estrogens: a model for hormonal carcinogenesis.

In order to study the effects of perinatal exposure to estrogens on the developing reproductive tract, outbred female mice were treated neonatally (days 1 to 5) with varying doses of diethylstilbestrol (DES) and sacrificed from 1 to 18 months of age. Uterine adenocarcinoma was observed in a time- and dose-related manner after DES treatment; at 18 months, neoplastic lesions were seen in 90% of the mice exposed neonatally to 2 micrograms/pup of DES/day, while none was observed in the corresponding control mice. These DES-induced uterine tumors were estrogen dependent; when DES-treated mice were ovariectomized before puberty, no uterine tumors developed. As a marker for neoplasia, uterine tumors were transplanted and carried as serial transplants in nude mice. The transplanted tissue retained some differentiated uterine gland structure and function and also required estrogen supplementation for maintenance. Additional groups of neonatal mice were treated with various DES analogues (hexestrol and tetrafluorodiethylstilbestrol) and steroidal estrogens. The compounds were ranked according to developmental estrogenic potency (hexestrol greater than trifluorodiethylstilbestrol greater than DES greater than 17 beta-estradiol). The combined prevalence of uterine atypical hyperplasia and adenocarcinoma follows the order of estrogenic potency. The experimental induction of these tumors will provide the basis for additional studies in mechanisms of hormonal carcinogenesis.

[Sex differences in the accumulation of estrogen receptors in nuclei of liver cells and increase of angiotensinogen concentration in the blood of rats after administration of low doses of synthetic estrogens]. / Polovye razlichiia v nakoplenii éstrogennykh retseptorov v iadrakh kletok pecheni i uvelichenii kontsentratsii angiotenzinogena v plazme krovi krys pri vvedenii niznikh doz sinteticheskikh éstrogenov.

The significance of sex differences in the level of estrogen receptors (ER) in hepatocytes for direct effects of estrogens in male and female rat livers was investigated. 4-5-fold increase in ER level and 20-30%-elevation in plasma angiotensinogen (AG) occurred after a single injection of 0.5 microgram of hexestrol (HE) in female and gonadectomized male rats. In male liver, where the cytosol ER content is two fold lower than that in female rats, nuclear ER level was shown to be very low and unchanged after HE injection, plasma AG also did not change. Injection of 0.5 microgram of ethinylestradiol produced similar effect. Injection of a greater dose of estrogen caused an enhancement in plasma AG level in males. The existence of sex differences in hepatic ER level seems to cause in some conditions different response of metabolic processes in male and female rat liver after estrogenization.

Effect of meso-hexestrol, a synthetic estrogen, on S-tubulin.

We have reported that meso-hexestrol, a synthetic estrogen, inhibits microtubule assembly and induces microtubule proteins into twisted ribbon structures. On the other hand, Serrano et al. proved that S-tubulin, which lacks the C-terminal moiety of tubulin subunits, assembles into sheet structures in the absence of microtubule-associated proteins (MAPs). In the present investigation, we attempted to clarify whether meso-hexestrol could induce the ribbon structure from S-tubulin. meso-Hexestrol delayed the initiation of polymerization of S-tubulin into sheet structures in a dose-dependent manner below 50 microM. But the effect of meso-hexestrol on S-tubulin was reduced in the presence of either tau or microtubule-associated protein 2 (MAP2) in a MAPs-concentration-dependent manner. At concentrations higher than 100 microM, meso-hexestrol inhibited the polymerization of S-tubulin into sheet structures, without forming ribbon structures. The present results may indicate that moso-hexestrol interacts with S-tubulin, and its interaction is affected by MAPs.

Hypotensive effects on spontaneously hypertensive rats and antifungal activity on various species of Fusarium oxysporum of diethylstilbestrol-related compounds.

The diethylstilbestrol-related compounds 3,3'-dihydroxy-alpha, beta-diethyldiphenylethane (I), diethylstilbestrol (II) and hexestrol (III) showed hypotensive effects on spontaneously hypertensive rats (SHR) and antifungal activities against all Fusarium oxysporum sp. tested. As previously reported, I had strong hypotensive action on normotensive rats at the dose of 10 mg/kg, while II and III showed weak hypotensive effects on these rats at the same dose. In this work, all three compounds also had hypotensive actions on SHR at the same dose. I showed the strongest hypotensive effect (-80.0 +/- 5.0 mmHg, 10 mg/kg, i.v.) on both SHR and normotensive rats. The three compounds also had antifungal activities against five kinds of Fusarium oxysporum sp. tested. Especially, II strongly inhibited the growth of Fusarium oxysporum f. sp. raphani IFO-9972 (minimum inhibitory concentration (MIC): 1.0 micrograms/ml).