Discovery and Optimization of a Compound Series Active against Trypanosoma cruzi, the Causative Agent of Chagas Disease.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. It is endemic in South and Central America and recently has been found in other parts of the world, due to migration of chronically infected patients. The current treatment for Chagas disease is not satisfactory, and there is a need for new treatments. In this work, we describe the optimization of a hit compound resulting from the phenotypic screen of a library of compounds against T. cruzi. The compound series was optimized to the level where it had satisfactory pharmacokinetics to allow an efficacy study in a mouse model of Chagas disease. We were able to demonstrate efficacy in this model, although further work is required to improve the potency and selectivity of this series.

Validation of Plasmodium falciparum dUTPase as the target of 5'-tritylated deoxyuridine analogues with anti-malarial activity.

BACKGROUND: Malaria remains as a major global problem, being one of the infectious diseases that engender highest mortality across the world. Due to the appearance of resistance and the lack of an effective vaccine, the search of novel anti-malarials is required. Deoxyuridine 5'-triphosphate nucleotido-hydrolase (dUTPase) is responsible for the hydrolysis of dUTP to dUMP within the parasite and has been proposed as an essential step in pyrimidine metabolism by providing dUMP for thymidylate biosynthesis. In this work, efforts to validate dUTPase as a drug target in Plasmodium falciparum are reported. METHODS: To investigate the role of PfdUTPase in cell survival different strategies to generate knockout mutants were used. For validation of PfdUTPase as the intracellular target of four inhibitors of the enzyme, mutants overexpressing PfdUTPase and HsdUTPase were created and the IC50 for each cell line with each compound was determined. The effect of these compounds on dUTP and dTTP levels from P. falciparum was measured using a DNA polymerase assay. Detailed localization studies by indirect immunofluorescence microscopy and live cell imaging were also performed using a cell line overexpressing a Pfdut-GFP fusion protein. RESULTS: Different attempts of disruption of the dut gene of P. falciparum were unsuccessful while a 3' replacement construct could recombine correctly in the locus suggesting that the enzyme is essential. The four 5'-tritylated deoxyuridine analogues described are potent inhibitors of the P. falciparum dUTPase and exhibit antiplasmodial activity. Overexpression of the Plasmodium and human enzymes conferred resistance against selective compounds, providing chemical validation of the target and confirming that indeed dUTPase inhibition is involved in anti-malarial activity. In addition, incubation with these inhibitors was associated with a depletion of the dTTP pool corroborating the central role of dUTPase in dTTP synthesis. PfdUTPase is mainly localized in the cytosol. CONCLUSION: These results strongly confirm the pivotal and essential role of dUTPase in pyrimidine biosynthesis of P. falciparum intraerythrocytic stages.

Preparation, structural characterization, and evaluation of ion-exchange behavior of a new polyoxometalate, [Me2NH2]3[Mo12O40S] in separation of carrier-free 90Y from 90Sr.

A new ion-exchanger having chemical formula [Me2NH2]3[Mo12O40S] and belonging to the class of Keggin type polyoxometalate was synthesized and characterized by single-crystal X-ray structure determination. The crystal of the exchanger is rhombohedral, space group R-3 with cell dimensions, a =16.504(18) Å, b =16.504(18)Å (1) Å, c =25.23(3)Å and α=90.00°, ß=90.00°, γ=120.00° and Z =6, 3.284gcm-3. The compound behaves as an ion-exchanger and it is significantly stable towards thermal, chemical environments and total radiation dose of 35.0kGy. Radiochemical separation of the short-lived daughter carrier-free 9°Y (T1/2 =64.08h) from its long-lived parent 90Sr (T1/2 =29 a) using this material at pH 6.0 with 1.0% EDTA solution as an eluent.

A ratiometric fluorescent chemosensor for iron: discrimination of Fe2+ and Fe3+ and living cell application.

A newly designed probe, 6-thiophen-2-yl-5,6-dihydrobenzo[4,5]imidazo-[1,2-c] quinazoline (HL(1)) behaves as a highly selective ratiometric fluorescent sensor for Fe(2+) at pH 4.0-5.0 and Fe(3+) at pH 6.5-8.0 in acetonitrile-HEPES buffer (1/4) (v/v) medium. A decrease in fluorescence at 412 nm and increase in fluorescence at 472 nm with an isoemissive point at 436 nm with the addition of Fe(2+) salt solution is due to the formation of mononuclear Fe(2+) complex [Fe(II)(HL)(ClO(4))(2)(CH(3)CN)(2)] (1) in acetonitrile-HEPES buffer (100 mM, 1/4, v/v) at pH 4.5 and a decrease in fluorescence at 412 nm and increase in fluorescence at 482 nm with an isoemissive point at 445 nm during titration by Fe(3+) salt due to the formation of binary Fe(3+) complex, [Fe(III)(L)(2)(ClO(4))(H(2)O)] (2) with co-solvent at biological pH 7.4 have been established. Binding constants (K(a)) in the solution state were calculated to be 3.88 × 10(5) M(-1) for Fe(2+) and 0.21 × 10(3) M(-1/2) for Fe(3+) and ratiometric detection limits for Fe(2+) and Fe(3+) were found to be 2.0 µM and 3.5 µM, respectively. The probe is a "naked eye" chemosensor for two states of iron. Theoretical calculations were studied to establish the configurations of probe-iron complexes. The sensor is efficient for detecting Fe(3+)in vitro by developing a good image of the biological organelles.

Development of a highly selective cell-permeable ratiometric fluorescent chemosensor for oxorhenium(V) ion.

A novel 6-(2-pyridinyl)-5,6-dihydrobenzimidazo[1,2-c]quinazoline (HL) serves as a first-time highly selective and sensitive ratiometric fluorescent chemosensor probe for oxorhenium (ReO(V)) ion in acetonitrile : water = 9 : 1 (v/v) at 25 °C. The decrease in fluorescence at 410 nm and increase in fluorescence at 478 nm with an isoemissive point at 444 nm in the presence of ReO(V) ion is accounted for by the formation of mononuclear [ReOL(2)Cl] complex, characterized by physico-chemical and spectroscopic tools. The fluorescence quantum yield of the chemosensor (HL) was only 0.198 at 410 nm, and it increased more than 3-fold in the presence of 2 equiv. of the ReO(V) ion at 478 nm. Interestingly, the introduction of other metal ions and relevant anions caused the fluorescence intensity at 478 nm to be either unchanged or weakened. The fluorescence-response fits a Hill coefficient of 2.088 indicates the formation of a 1 : 2 stoichiometry for the L-ReO(V) complex. In the concentration range of 0-20 µM of oxorhenium(V) species calibration graph was linear with correlation coefficient (R) of 0.99994 and the calibration sensitivity was found to be 4.0 × 10(-7) M. The cellular image in the confocal microscope clearly indicated the presence of ReO(V) in Candida albicans cells using this chemosensor (HL).

A highly selective fluorescent chemosensor for zinc ion and imaging application in living cells.

A new 2,6-bis(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-4-methylphenol (1) serves as a highly selective and sensitive fluorescent probe for Zn(2+) in a HEPES buffer (50 mM, DMSO:water = 1:9 (v/v), pH = 7.2) at 25 °C. The increase in fluorescence in the presence of Zn(2+) is accounted for by the formation of dinuclear Zn(2+) complex [Zn(2)(C(35)H(25)N(6)O)(OH)(NO(3))(2)(H(2)O)] (2), characterized by X-ray crystallography. The fluorescence quantum yield of the chemosensor 1 is only 0.019, and it increases more than 12-fold (0.237) in the presence of 2 equiv of the zinc ion. Interestingly, the introduction of other metal ions causes the fluorescence intensity to be either unchanged or weakened. By incubation of cultured living cells (A375 and HT-29) with the chemosensor 1, intracellular Zn(2+) concentrations could be monitored through selective fluorescence chemosensing.

A new water-soluble copper(II) complex as a selective fluorescent sensor for azide ion.

{[Cu(2)(H(2)L)(OH)(H(2)O)].(ClO(4))(2)(H(2)O)} (1) serves as a selective azide ion fluorescent sensor in aqueous medium. 1 binds with N(3)(-) to give [Cu(6)(HL)(2)(mu(1,1)-N(3))(6)] (2) which imposes rigidity and decreases the non-radiative decay of the excited state to give rise to fluorescent enhancement. 1 is highly selective over other various anions in aqueous medium.

A novel aluminum vanadate ion exchanger and its use for separation of (137m)Ba, (115m)In and (110m)Ag from (137)Cs, (115)Cd and (198)Au, respectively.

A newly designed inorganic ion exchanger, based on aluminum vanadate, has been synthesized and characterized by elemental analysis, spectroscopic tools and powdered X-ray diffraction. The insoluble poorly polycrystalline material is highly stable towards thermal and radiation doses and in various chemical environments. The data of exchange capacities of the solid material for the different alkali and alkaline metal ions determined by batch technique show that the compound can be employed as an ion exchanger. The successful radiochemical separations of the no carrier added daughter nuclides; (137m)Ba and (115m)In from their respective parents present in equilibrium mixtures have been carried out using this material. Elutions of (137m)Ba and (115m)In were performed using 0.0426molL(-1) ascorbic acid solution and 4.0molL(-1) HCl, respectively, after sorption of the equilibrated mixtures (137)Cs-(137m)Ba at 0.01molL(-1) HCl medium and (115)Cd-(115m)In at pH 7.0, respectively. In another column operation, it has been observed that the separation of gold and silver is possible with the help of the eluents, 0.01% alcoholic solution of Rhodamine-B for gold and 0.5% thiourea solution in 0.1molL(-1) HClO(4) for silver, respectively, after the sorption of no carrier added onto this material at pH 2.0, at a no carrier added level.