Kinetics of Flavoenzyme-Catalyzed Reduction of Tirapazamine Derivatives: Implications for Their Prooxidant Cytotoxicity.

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit promising antibacterial, antiprotozoal, and tumoricidal activities. Their action is typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the mechanism(s) of aerobic mammalian cell cytotoxicity of ArN→O performing the parallel studies of their reactions with NADPH:cytochrome P-450 reductase (P-450R), adrenodoxin reductase/adrenodoxin (ADR/ADX), and NAD(P)H:quinone oxidoreductase (NQO1); we found that in P-450R and ADR/ADX-catalyzed single-electron reduction, the reactivity of ArN→O (n = 9) increased with their single-electron reduction midpoint potential (E17), and correlated with the reactivity of quinones. NQO1 reduced ArN→O at low rates with concomitant superoxide production. The cytotoxicity of ArN→O in murine hepatoma MH22a and human colon adenocarcinoma HCT-116 cells increased with their E17, being systematically higher than that of quinones. The cytotoxicity of both groups of compounds was prooxidant. Inhibitor of NQO1, dicoumarol, and inhibitors of cytochromes P-450 α-naphthoflavone, isoniazid and miconazole statistically significantly (p < 0.02) decreased the toxicity of ArN→O, and potentiated the cytotoxicity of quinones. One may conclude that in spite of similar enzymatic redox cycling rates, the cytotoxicity of ArN→O is higher than that of quinones. This is partly attributed to ArN→O activation by NQO1 and cytochromes P-450. A possible additional factor in the aerobic cytotoxicity of ArN→O is their reductive activation in oxygen-poor cell compartments, leading to the formation of DNA-damaging species similar to those forming under hypoxia.

Production of the recombinant antimicrobial peptide UBI18-35 in Escherichia coli.

Radiolabeled peptides derived from ubiquicidine (UBI) are of great interest for early and highly accurate scintigraphic detection and differentiation of infection and sterile inflammation. In the present work the recombinant antimicrobial peptide UBI18-35 - a fragment of the human natural cationic peptide ubiquicidine - was produced in Escherichia coli for the first time. The insoluble expression of the peptide in fusion with ketosteroid isomerase provided high yield, about 6 mg of UBI18-35 per liter. We developed an approach to produce the antimicrobial peptide UBI18-35, that encompasses inclusion body isolation and size exclusion chromatography. This method could be the basis for industrial biotechnological production of diagnostic system components that are in high demand.

Computer-aided design of aptamers for cytochrome p450.

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M.

Synthesis, physico-chemical properties and effect of adenosine thiamine triphosphate on vitamin B1 metabolism in the liver of alloxan diabetic rats.

BACKGROUND: Adenosine thiamine triphosphate (AThTP) is a nucleotide discovered in bacteria and some other living organisms more than a decade ago. No biochemical function for AThTP has been established yet, however, experimental data available indicate its possible involvement in metabolic regulation or cell signaling. Metabolism of AThTP in mammals, as well as the feasibility of its pharmacological application, is essentially unstudied. METHODS: Preparative low-pressure chromatography was employed to purify chemically synthesized AThTP with its further analysis by mass spectrometry, HPLC, UV and fluorescence spectroscopy. Enzyme activity assays along with HPLC were used to examine the effects of AThTP and thiamine on vitamin B1 metabolism in the liver of alloxan-induced diabetic rats. RESULTS: An improved procedure for AThTP synthesis and purification is elaborated. Solution stability, optical spectral properties and the molar absorption coefficient for AThTP were determined. The levels of thiamine compounds were found to be increased in the liver of diabetic rats. Neither AThTP nor thiamine treatment affected hepatic vitamin B1 metabolism. Fasting blood glucose concentration was also unchangeable after AThTP or thiamine administration. GENERAL SIGNIFICANCE: Contrast to the widespread view about thiamine deficiency in diabetes, our results clearly shows an adaptive increase in the level of B1 vitamers in the liver of alloxan diabetic rats with no further rising after AThTP or thiamine treatment at a moderate dose. Neither AThTP nor thiamine is effective in glycaemic control. These findings are to be considered in future studies dealing with thiamine or its analogues application to correct metabolic disturbances in diabetes.

5Flavoenzyme-catalyzed single-electron reduction of nitroaromatic antiandrogens: implications for their cytotoxicity.

The therapeutic action of nitroaromatic antiandrogens nilutamide and flutamide may be complicated by their cytotoxicity, whose mechanisms are still incomprehensively understood. In particular this concerns the enzymatic redox cycling of flutamide and its metabolites, and its impact on their cytotoxicity. In this work, we examined the single-electron reduction of nilutamide, flutamide, its metabolites 2-hydroxyflutamide and 4-nitro-3-trifluorormethyl-phenylamine, and a topical antiandrogen (3-amino-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl)-phenyl) propanamide by NADPH:cytochrome P-450 reductase and adrenodoxin reductase/adrenodoxin. The obtained steady-state bimolecular rate constants of oxidant reduction (kcat/Km) enabled to establish single-electron reduction midpoint potentials (E17) of compounds, -0.377 - -0.413 V, which were in line with enthalpies of formation of their free radicals, obtained by quantum mechanical calculations. Using murine hepatoma MH22a cells, the obtained cytotoxicity vs. E17 correlation based on the data of model nitroaromatic compounds shows that redox cycling and oxidative stress could be the main factor of cytotoxicity of nitroaromatic antiandrogens. Other minor cytotoxicity factors could be their redox metabolism involving NAD(P)H:quinone oxidoreductase (NQO1) and cytochromes P-450.