Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy.

BACKGROUND: The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool. METHODS: Vcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice. RESULTS: 5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 µM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals. CONCLUSIONS: We have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy.

Flucytosine analogues obtained through Biginelli reaction as efficient combinative antifungal agents.

Invasive fungal infection is a problem that continues to challenge the healthcare sector. New antifungals and new therapeutic strategies are needed to address this challenge. We previously reported that the combination of a synthetic compound with a drug with known mechanism of action is a good strategy to treat aggressive and resistant fungi. Here we revisited our approach and synthesized structural analogues of flucytosine, which is a synthetic antifungal and is being studied for its use in combination therapy with other antifungal drugs. Pyrimidin-one and -thione (often known as DHPM's) as flucytosine analogues were obtained through a Biginelli reaction of corresponding aldehydes, ethylacetoacetate and urea/thiourea. Structure was confirmed by FTIR, 1HNMR, 13CNMR, COSY and MS (ESI+) analysis. All the newly synthesized derivatives were evaluated for the antifungal activity alone and in combination of two most commonly used antifungal drugs, amphotericin B and fluconazole against different clinically isolated Candida albicans strains. Minimum inhibitory concentration results confirmed that BG4 possess high antifungal activity against all the tested strains (MIC = 1-32 µg/ml). For all the combinations with amphotericin B and fluconazole, 37% were synergistic followed by 30% additive and 24% indifferent interactions. Interestingly, 9% antagonistic interaction was observed when BG1 and BG3 were combined with fluconazole, however, no antagonistic interaction was observed with amphotericin B. In-depth studies of all the synergies were done by constructing isobolograms with nine different ratio combinations. These results warrant the use of DHPM derivatives as chemosensitising agents which could lower down the dosages of the antifungal drugs to treat invasive fungal diseases.

Tridentate ionic hydrogen-bonding interactions of the 5-fluorocytosine cationic dimer and other 5-fluorocytosine analogues characterized by IRMPD spectroscopy and electronic structure calculations.

Ionic hydrogen-bonding interactions have been found in several clusters formed by 5-fluorocytosine (5-FC). The chloride and trimethylammonium cluster ions, along with the cationic (proton-bound) dimer have been characterized by infrared multiple-photon dissociation (IRMPD) spectroscopy and electronic structure calculations performed at the B2PLYP/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. IRMPD action spectra, in combination with calculated spectra and relative energetics, indicate that it is most probable that predominantly a single isomer exists in each experiment. For the 5-FC-trimethylammonium cluster specifically, the calculated spectrum of the lowest-energy isomer convincingly matches the experimental spectrum. Interestingly, the cationic dimer of 5-FC was found to have a single energetically relevant isomer (Cationic-IV) involving a tridentate ionic hydrogen-bonding interaction. The three sites of intermolecular ionic hydrogen bonds in this isomer interact very efficiently, leading to a significant calculated binding energy of 180 kJ/mol. The magnitude of the calculated binding energy for this species, in combination with the strong correlation between the simulated and IRMPD spectra, suggests that a tridentate-proton-bound dimer was observed predominantly in the experiments. Comparison of the calculated relative Gibbs free energies (298 K) for this species and several of the other isomers considered also supports the likelihood of the dominant protonated dimer existing as Cationic-IV.

Synthesis and biological activity of the new 5-fluorocytosine derivatives, 5'-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5'-carboxylic acid.

A series of 5-fluorocytosine derivatives, 5'-deoxy-N-alkyloxycarbonyl-5-fluorocytosine-5'-carboxylic acid 6, were synthesized and evaluated for their antitumor activity.

Stereospecific synthesis and biological evaluations of beta-L-pentofuranonucleoside derivatives of 5-fluorouracil and 5-fluorocytosine.

In the search for new chemotherapeutic agents, we have focused our work on the synthesis and the study of several unnatural beta-L-nucleoside analogues. In this paper, we report on the synthesis of beta-L-pentofuranonucleosides (and their 2'-deoxy derivatives) of 5-fluorouracil and their inhibitory effects on the proliferation of several murine and human tumor cells. The corresponding 5-fluorocytosine derivatives were also synthesized and their anti-HIV and anti-HBV activities have been evaluated.

Structure and conformation of N4-hydroxycytosine and N4-hydroxy-5-fluorocytosine. A theoretical ab initio study.

Optimal molecular geometries and molecular energies were obtained for N4-hydroxycytosine and its 5-fluoro congener with the use of the theoretical ab initio quantum mechanical calculations within the Self Consistent Field method corrected for the electron correlation effects by the second-order Many Body Perturbation Theory (SCF + MBPT(2)). The 6-31G Gaussian basis set was employed. Several tautomeric and rotameric forms were considered. For N4-hydroxycytosine and N4-hydroxy-5-fluorocytosine the imino tautomer (in the conformation syn relatively to the N3-nitrogen atom) appeared to be the most stable form. The imino tautomer of N4-hydroxy-cytosine in the anti rotameric form is by 12.8 kJ mol-1 less stable than the imino-syn form. The 5-fluoro substituent raises the energy difference between the syn and anti rotamers up to 38.5 kJ mol-1. The potential energy barrier for the syn-anti rotation in the imino form of N4-hydroxycytosine is estimated to be about 180 kJ/mol. The results presented in this paper suggest that the syn-imino and anti-imino forms can be treated as two structural isomers that do not interconvert at temperatures relevant to biochemical conditions. The theoretical results also show that the amino tautomeric forms do not compete with the imino forms in the gas-phase and in non-polar and weakly-polar environment. In a polar environment (e.g., in aqueous solutions), however, one may expect an increased population of the amino forms. Qualitatively, the results of the present study agree well with the available experimental and theoretical data for N4-hydroxycytosine and some of its derivatives. The implications of the present study are discussed in relation to the molecular mechanisms of mutagenesis caused by NH2OH and of enzyme (thymidylate synthase) inhibition by N4-hydroxydeoxycytidine monophosphate.

Flucytosine conversion to fluorouracil in humans: does a correlation with gut flora status exist? A report of two cases using fluorine-19 magnetic resonance spectroscopy.

A relationship between the gut flora level, particularly gram-negative enterobacilli, and the in vivo flucytosine conversion to fluorouracil has been observed in humans from the fluorine-19 magnetic resonance spectroscopy analysis of urine from two patients treated with the flucytosine- (6 to 9 g/day) amphotericin B (1 mg/kg/day) combination. Indeed the percentage of fluorouracil metabolites was extremely low (less than 0.6% of total fluorinated compounds excreted) when the number of enterobacillary colonies was low (less than 10(3] and higher (3.5 to 8.8%) when enterobacillary colonies were under reconstitution or in the normal range (10(5) to 10(8]. The intestinal microflora assessment may therefore be of high interest to predict the risk of an additive flucytosine-induced myelotoxicity suspected to be due to fluorouracil during flucytosine chronic therapy.

Anticandidal activity of 5-fluorocytosine-peptide conjugates.

An approach to the development of new anticandidal drugs is described that employs peptides as carriers of toxic agents into cells. 5-Flurorcytosine (5-FC) was chosen as a toxic agent with which to prepare 5-FC-peptide conjugates as models to test the carrier proposal. Model compounds were synthesized and then tested for antiyeast activity against S. Cerevisiae 9763, C. albicans 1-V, C. albicans WD 18-4, and C. Krusei 1-T. The 5-FC derivatives showed antiyeast activity comparable to 5-FC in all strains except C. krusei 1-T, in which case the compounds were less active. The solution stabilities of 5-FC conjugates at 37 degrees C were tested in the same growth medium used for susceptibility testing. The results indicated a range of stabilities where the half-life (t1/2) = 0.3--17.6 h. These results and those obtained in the susceptibility testing suggest extracellular hydrolysis and indicate that the type of linkage used to conjugate 5-FC to peptides will not provide appropriate compounds to evaluate the peptide-carrier concept.

Biological consequences of incorporation of 5-fluorocytidine in the RNA of 5-fluorouracil-treated eukaryotic cells.

Treatment of HeLa cells with 5-fluoro-[3H]uracil leads to the incorporation into cellular RNA of 5-fluorocytidine to the extent of about 0.2% of the 5-fluorouridine incorporated. In tobacco mosaic virus RNA produced in tobacco leaves this ratio is one order of magnitude lower. Copolymers of cytidylic with 5-fluorocytidylic acids show unchanged template activity with E. coli RNA polymerase, but slightly altered messenger activity in the wheat germ system, compared to poly(C), and it is suggested that some of the biological consequences of 5-fluorouracil treatment of living cells and organisms may be attributed to this mechanism.