Therapeutic candidates for the Zika virus identified by a high-throughput screen for Zika protease inhibitors.

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Methacycline displays a strong efficacy in reducing toxicity in a SCA3 Caenorhabditis elegans model.

BACKGROUND: We have previously demonstrated the neuroprotective activity of tetracycline on a Spinocerebellar Ataxia 3 nematode model. Here, we present the screening of a small library of tetracycline congeners in order to identify the most effective compound in preventing ataxin-3 aggregation. METHODS: We performed the assays on the Josephin Domain as it is directly involved in the onset of fibrillation. We used thioflavin T and solubility assays to spot out the most effective tetracycline congeners; Fourier transform infrared and NMR spectroscopies to characterize their mode of action. We employed an ataxic Caenorhabditis elegans model to evaluate the pharmacological efficacy of tetracycline congeners. RESULTS: Methacycline was identified as the most effective compound. Like tetracycline, methacycline neither significantly affected the aggregation kinetics nor did it change the secondary structures of the final aggregates but increased the solubility of the aggregated species. Saturation transfer NMR experiments demonstrated methacycline capability to only bind the oligomeric species of Josephin Domain. Competition assays also showed that methacycline binds to the Josephin Domain more tightly than tetracycline. The treatment with methacycline induced a significant improvement in motility and locomotion of the transgenic C. elegans without changing its lifespan. The efficacy was distinctly stronger than that of tetracycline. Noteworthy, unlike tetracycline, methacycline was able to retard aging-related decline in motility of even the healthy worms used. CONCLUSIONS: The apparent absence of toxic effects displayed by methacycline, along with its stronger efficacy in contrasting expanded ataxin-3 toxicity, makes it a possible candidate for a chronic treatment of the disease.

Inhibition of epithelial-to-mesenchymal transition and pulmonary fibrosis by methacycline.

A high-throughput small-molecule screen was conducted to identify inhibitors of epithelial-mesenchymal transition (EMT) that could be used as tool compounds to test the importance of EMT signaling in vivo during fibrogenesis. Transforming growth factor (TGF)-ß1-induced fibronectin expression and E-cadherin repression in A549 cells were used as 48-hour endpoints in a cell-based imaging screen. Compounds that directly blocked Smad2/3 phosphorylation were excluded. From 2,100 bioactive compounds, methacycline was identified as an inhibitor of A549 EMT with the half maximal inhibitory concentration (IC50) of roughly 5 µM. In vitro, methacycline inhibited TGF-ß1-induced α-smooth muscle actin, Snail1, and collagen I of primary alveolar epithelial cells . Methacycline inhibited TGF-ß1-induced non-Smad pathways, including c-Jun N-terminal kinase, p38, and Akt activation, but not Smad or ß-catenin transcriptional activity. Methacycline had no effect on baseline c-Jun N-terminal kinase, p38, or Akt activities or lung fibroblast responses to TGF-ß1. In vivo, 100 mg/kg intraperitoneal methacycline delivered daily beginning 10 days after intratracheal bleomycin improved survival at Day 17 (P < 0.01). Bleomycin-induced canonical EMT markers, Snail1, Twist1, collagen I, as well as fibronectin protein and mRNA, were attenuated by methacycline (Day 17). Methacycline did not attenuate inflammatory cell accumulation or alter TGF-ß1-responsive genes in alveolar macrophages. These studies identify a novel inhibitor of EMT as a potent suppressor of fibrogenesis, further supporting the concept that EMT signaling is important to lung fibrosis. The findings also provide support for testing the impact of methacycline or doxycycline, an active analog, on progression of human pulmonary fibrosis.

[Sensitivity of Pseudomonas mallei to tetracyclines and their effectiveness in experimental glanders]. / Chuvstvitel'nost' Pseudomonas mallei k tetratsiklinam i ikh éffektivenost' pri éksperimental'nom sape.

MICs and MBcCs of minocycline, doxycycline, methacycline and chlortetracycline for 8 strains of Pseudomonas mallei were determined. The chemotherapeutic efficacy of minocycline and doxycycline was studied on golden hamsters and their efficacy indices were estimated in comparison with those of chlortetracycline in the prophylaxis and treatment of experimental malleus. Minocycline was shown to be the most efficient drug in the treatment of malleus. Doxycycline in a dose of 0.25 mg/kg practically had the same efficacy as chlortetracycline in a dose of 25 mg/kg. Methacycline was inefficient.

[Combined action of amphotericin B and methacyclin on Candida albicans]. / Sochetannoe deistvie amfoteritsina B i metatsiklina na kletki Candida albicans.

The combined effect of amphotericin B, a polyene antibiotic, and metacycline, a tetracycline antibiotic, on the cells of C. albicans was studied. The method of square titration followed by quantitative plating of the samples was used for estimation of the combination efficiency. An attempt was also made to investigate the characteristic features of metacycline penetration into the yeast cells under the effect of various doses of the polyene antibiotic. The capacity of metacycline for fluorescence in the yellow-green pectral region was employed for this purpose. It was shown that the drugs had a synergistic effect on C. albicans. The fluorescence research methods allowed one to demonstrate that even low subinhibitory doses of amphotericin B increased the permeability level of the cytoplasmic membrane and provided penetration of metacycline into the cytoplasm almost during the first hours of the contact. The time course of metacycline cumulation in the cells was followed up and the characteristic features of the antibiotic localization were analysed.

[Fluorimetric study of methacycline complex formation with beryllium, aluminum, magnesium, calcium and zinc ions]. / Fluorimetricheskoe izuchenie kompleksoobrazovaniia metatsiklina s ionami berilliia, aliuminiia, magniia, kal'tsiia i tsinka.

Complex formation of metacycline (Pv) with beryllium, aluminium, magnesium, calcium and zinc ions in aqueous solutions was studied fluorometrically. The antibiotic formed the following compounds: CaPv2 (pH 9.45), MgPv (pH 10.40), Be Pv (pH 7.0 and 11), Ca Pv (pH 13.5), Zn Pv (pH 7.15) and Al Pv (pH 6.0). Relationship between the solution fluorescence level and the values of pH, the quantum yield of fluorescence of Pv and the complexes and the kinetics of the reactions of complex formation of Pv with Be and Al were studied. The mechanism of Pv complex formation with the metal ions is discussed. A method for analysis of microgram amounts of Pv in biological fluids was developed. The method is based on the antibiotic fluorescent reaction with magnesium ions.

[Comparative evaluation of methacycline hydrochloride capsules and tablets according to their dissolubility and rate of solution]. / Sravnitel'naia otsenka kapsul i tabletok metatsiklina gidrokhlorida po raspadeamosti i skorosti rastvoreniia.

Desintegration and dissolution of capsules and tablets of methacycline hydrochloride were studied. The study on solubility of methacycline hydrochliride capsules filled with methacycline granulate or powder according to the same formula showed that the rate of the antibiotic liberation from the capsules filled with the powder decreased during storage while that from the capsules filled with the granulate did not change. Investigation of the effect of the mass packing value in a drop on the antibiotic liberation from the capsules showed that an increase in the packing coefficient above 1.38 resulted in a marked decrease in the rate of methacycline liberation from the capsules filled with the granulate. No correlation between desintegration and dissolution of methacycline capsules and tablets was found.

[Metacycline, its chemotherapeutic activity and pharmacokinetic characteristics]. / Metatsiklin--khimioterapevticheskaia aktivnost' i osobennosti farmakokinetiki

Methacycline prepared at the All-Union Research Institute of Antibiotics was characterized by high activity against staphylococci and gramnegative bacteria. A somewhat higher antistaphylococcal activity of methacycline as compared to oxytetracycline was found. The concentrations of methacycline in the kidneys, liver and lungs corresponded to its levels in the blood and were 3 times higher than those of oxytetracycline administered in close doses. Methacycline was characterized by a higher chemotherapeutic activity as compared to oxytetracycline in the treatment of staphylococcal pneumonia. The values of CD50 of methacycline were 1.5 times lower than those of oxytetracycline.

The effect of minocycline on Candida albicans.

Minocycline, a new tetracycline derivative, was found to inhibit the growth of Candida albicans. Inhibition was much affected by the composition of the medium and was difficult to demonstrate in fluid cultures. Study of the rate of budding in shallow broth cultures in Petri dishes showed that the addition of 20 mug/ml minocycline prolonged the lag phase by three hours. C. tropicalis was similarly inhibited and C. guilliermondii and C. parapsilosis to a lesser degree. Six other tetracyclines were tested and found to inhibit Candida only in very high concentrations.