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.

Modified bile acids and androstanes-Novel promising inhibitors of human cytochrome P450 17A1.

Cytochromes P450 are key enzymes for steroid hormone biosynthesis in human body. They are considered as targets for the screening of novel high efficient drugs. The results of screening of bile acids and androstane derivatives toward human recombinant steroid 17α-hydroxylase/17,20-lyase (CYP17A1) are presented in this paper. A group of steroids, binding with micromolar or submicromolar affinity (in a range from 9 µM - less than 0.1 µM), was identified. Results presented here showed that these steroidal compounds are able to decrease rate of hydroxylation of essential CYP17A1 substrate - progesterone, while some compounds completely inhibited enzyme activity. Structure-activity relationship (SAR) analysis based on in vitro and in silico studies showed that high affinity of the enzyme to bile acids derivatives is correlated with side chain hydrophobicity and presence of hydroxyl or keto group at C3 position. From the other side, bile acid-derived compounds with more polar side chain or substituents at C7 and C12 positions possess higher Kd values. Among androstane-derived steroids couple of Δ5-steroids with hydroxyl group at C3 position, as well as 16,17-secosteroids, were found to be high affinity ligands of this enzyme. The data obtained could be useful for the design of novel highly efficient inhibitors of CYP17A1, since the bile acids-derived compounds are for first time recognized as effective CYP17A1 inhibitors.

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.

Evidence That Compound I Is the Active Species in Both the Hydroxylase and Lyase Steps by Which P450scc Converts Cholesterol to Pregnenolone: EPR/ENDOR/Cryoreduction/Annealing Studies.

Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22(R)-hydroxycholesterol (HC) and then 20α,22(R)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20-C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) J. Am. Chem. Soc. 134, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170-180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and (1)H ENDOR measurements of the primary product formed in D2O demonstrate that Cpd I is the active species. They show that the primary product contains Fe(III) coordinated to the 20-O(1)H of DHC with the (1)H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone.

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.