RESUMEN
American trypanosomiasis is a worldwide health problem that requires attention due to ineffective treatment options. We evaluated n-butyl and isobutyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives against trypomastigotes of the Trypanosoma cruzi strains NINOA and INC-5. An in silico analysis of the interactions of 1,4-di-N-oxide on the active site of trypanothione reductase (TR) and an enzyme inhibition study was carried out. The n-butyl series compound identified as T-150 had the best trypanocidal activity against T. cruzi trypomastigotes, with a 13% TR inhibition at 44 µM. The derivative T-147 behaved as a mixed inhibitor with Ki and Ki' inhibition constants of 11.4 and 60.8 µM, respectively. This finding is comparable to the TR inhibitor mepacrine (Ki = 19 µM).
Asunto(s)
Enfermedad de Chagas , Tripanocidas , Trypanosoma cruzi , Humanos , Tripanocidas/farmacología , Tripanocidas/química , Quinoxalinas/química , Óxidos/farmacología , NADH NADPH Oxidorreductasas , Enfermedad de Chagas/tratamiento farmacológico , Inhibidores Enzimáticos/químicaRESUMEN
SIGNIFICANCE: Glutaredoxins are ubiquitous small thiol proteins of the thioredoxin-fold superfamily. Two major groups are distinguished based on their active sites: the dithiol (2-C-Grxs) and the monothiol (1-C-Grxs) glutaredoxins with a CXXC and a CXXS active site motif, respectively. Glutaredoxins are involved in cellular redox and/or iron sulfur metabolism. Usually their functions are closely linked to the glutathione system. Trypanosomatids, the causative agents of several tropical diseases, rely on trypanothione as principal low molecular mass thiol, and their glutaredoxins readily react with the unique bis(glutathionyl) spermidine conjugate. RECENT ADVANCES: Two 2-C-Grxs and three 1-C-Grxs have been identified in pathogenic trypanosomatids. The 2-C-Grxs catalyze the reduction of glutathione disulfide by trypanothione and display reductase activity towards protein disulfides, as well as protein-glutathione mixed disulfides. In vitro, all three 1-C-Grxs as well as the cytosolic 2-C-Grx of Trypanosoma brucei can complex an iron-sulfur cluster. Recently the structure of the 1-C-Grx1 has been solved by NMR spectroscopy. The structure is very similar to those of other 1-C-Grxs, with some differences in the loop containing the conserved cis-Pro and the surface charge distribution. CRITICAL ISSUES: Although four of the five trypanosomal glutaredoxins proved to coordinate an iron-sulfur cluster in vitro, the physiological role of the mitochondrial and cytosolic proteins, respectively, has only started to be unraveled. FUTURE DIRECTIONS: The use of trypanothione by the glutaredoxins has established a novel role for this parasite-specific dithiol. Future work should reveal if these differences can be exploited for the development of novel antiparasitic drugs.
Asunto(s)
Glutarredoxinas/fisiología , Glutatión/análogos & derivados , Proteínas Protozoarias/fisiología , Espermidina/análogos & derivados , Trypanosoma/enzimología , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Glutarredoxinas/química , Glutatión/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Conformación Proteica , Proteínas Protozoarias/química , Espermidina/metabolismo , Tripanosomiasis/parasitologíaRESUMEN
AIMS: Monothiol glutaredoxins (1-C-Grxs) are small proteins linked to the cellular iron and redox metabolism. Trypanosoma brucei brucei, model organism for human African trypanosomiasis, expresses three 1-C-Grxs. 1-C-Grx1 is a highly abundant mitochondrial protein capable to bind an iron-sulfur cluster (ISC) in vitro using glutathione (GSH) as cofactor. We here report on the functional and structural analysis of 1-C-Grx1 in relation to its ISC-binding properties. RESULTS: An N-terminal extension unique to 1-C-Grx1 from trypanosomatids affects the oligomeric structure and the ISC-binding capacity of the protein. The active-site Cys104 is essential for ISC binding, and the parasite-specific glutathionylspermidine and trypanothione can replace GSH as the ligands of the ISC. Interestingly, trypanothione forms stable protein-free ISC species that in vitro are incorporated into the dithiol T. brucei 2-C-Grx1, but not 1-C-Grx1. Overexpression of the C104S mutant of 1-C-Grx1 impairs disease progression in a mouse model. The structure of the Grx-domain of 1-C-Grx1 was solved by nuclear magnetic resonance spectroscopy. Despite the fact that several residues--which in other 1-C-Grxs are involved in the noncovalent binding of GSH--are conserved, different physicochemical approaches did not reveal any specific interaction between 1-C-Grx1 and free thiol ligands. INNOVATION: Parasite Grxs are able to coordinate an ISC formed with trypanothione, suggesting a new mechanism of ISC binding and a novel function for the parasite-specific dithiol. The first 3D structure and in vivo relevance of a 1-C-Grx from a pathogenic protozoan are reported. CONCLUSION: T. brucei 1-C-Grx1 is indispensable for mammalian parasitism and utilizes a new mechanism for ISC binding.
Asunto(s)
Mitocondrias/enzimología , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Coenzimas/química , Secuencia de Consenso , Cisteína/química , Femenino , Glutarredoxinas/química , Glutatión/química , Proteínas Hierro-Azufre/química , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Parasitemia/parasitología , Unión Proteica , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/parasitologíaRESUMEN
New benzofuroxans were developed and studied as antiproliferative Trypanosoma cruzi agents. Compounds displayed remarkable in vitro activities against different strains, Tulahuen 2, CL Brener and Y. Its unspecific cytotoxicity was evaluated using human macrophages being not toxic at a concentration at least 8 times, and until 250 times, that of its T. cruzi IC50. Some biochemical pathways were studied, namely parasite respiration, cysteinyl active site enzymes and reaction with glutathione, as target for the mechanism of action. Not only T. cruzi respiration but also Cruzipain or trypanothione reductase were not affected, however the most active derivatives, the vinylsulfinyl- and vinylsulfonyl-containing benzofuroxans, react with glutathione in a redox pathway. Furthermore, the compounds showed good in vivo activities when they were studied in an acute murine model of Chagas' disease. The compounds were able to reduce the parasite loads of animals with fully established T. cruzi infections.
Asunto(s)
Benzoxazoles/síntesis química , Enfermedad de Chagas/tratamiento farmacológico , Sulfonas/síntesis química , Tripanocidas/síntesis química , Trypanosoma cruzi/efectos de los fármacos , Compuestos de Vinilo/síntesis química , Animales , Anticuerpos Antiprotozoarios/sangre , Benzoxazoles/farmacología , Benzoxazoles/toxicidad , Línea Celular , Cisteína Endopeptidasas/metabolismo , Femenino , Glutatión/metabolismo , Humanos , Macrófagos/efectos de los fármacos , Ratones , Modelos Moleculares , NADH NADPH Oxidorreductasas/metabolismo , Oxidación-Reducción , Consumo de Oxígeno/efectos de los fármacos , Proteínas Protozoarias , Estereoisomerismo , Relación Estructura-Actividad , Sulfonas/química , Sulfonas/farmacología , Tripanocidas/farmacología , Tripanocidas/toxicidad , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/fisiología , Compuestos de Vinilo/química , Compuestos de Vinilo/farmacologíaRESUMEN
In the search for new therapeutic tools against American Trypanosomiasis palladium complexes with bioactive nitrofuran-containing thiosemicarbazones as ligands were obtained. Sixteen novel palladium (II) complexes with the formulas [PdCl2(HL)] and [Pd(L)2] were synthesized, and the crystal structure of [Pd(5-nitrofuryl-3-acroleine thiosemicarbazone)2] x 3DMSO was solved by X-ray diffraction methods. Most complexes showed higher in vitro growth inhibition activity against Trypanosoma cruzi than the standard drug Nifurtimox. In most cases, the activity of the ligand was maintained or even increased as a result of palladium complexation. In addition, the complexes' mode of antitrypanosomal action was investigated. Although the complexes showed strong DNA binding, all data strongly suggest that the main trypanocidal mechanism of action is the production of oxidative stress as a result of their bioreduction and extensive redox cycling. Moreover, the complexes were found to be irreversible inhibitors of trypanothione reductase.
Asunto(s)
ADN/química , Nitrofuranos/síntesis química , Compuestos Organometálicos/síntesis química , Paladio , Tiosemicarbazonas/síntesis química , Tripanocidas/síntesis química , Animales , Cristalografía por Rayos X , Dimetilsulfóxido/química , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres/metabolismo , Ligandos , Espectroscopía de Resonancia Magnética , Estructura Molecular , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Nitrofuranos/química , Nitrofuranos/farmacología , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Oxidación-Reducción , Estrés Oxidativo , Espectroscopía Infrarroja por Transformada de Fourier , Espectrometría Raman , Relación Estructura-Actividad , Tiosemicarbazonas/química , Tiosemicarbazonas/farmacología , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/metabolismoRESUMEN
Phenothiazine cation radicals (PTZ+*) irreversibly inactivated Trypanosoma cruzi dihydrolipoamide dehydrogenase (LADH). These radicals were obtained by phenothiazine (PTZ) peroxidation with myeloperoxidase (MPO) or horseradish peroxidase (HRP/H2O2) systems. LADH inactivation depended on PTZ structure and incubation time. After 10 min incubation of LADH with the MPO-dependent systems, promazine, trimeprazine and thioridazine were the most effective; after 30 min incubation, chlorpromazine, prochlorperazine and promethazine were similarly effective. HRP-dependent systems were equally or more effective than the corresponding MPO-dependent ones. Chloro, trifluoro, propionyl and nitrile groups at position 2 of the PTZ ring significantly decreased molecular activity, specially with the MPO/H2O2 systems. Comparison of inactivation values for LADH and T. cruzi trypanothione reductase demonstrated a greater sensitivity of LADH to chlorpromazine and perphenazine and a 10-fold lower sensitivity to promazine, thioridazine and trimeprazine. Alkylamino, alkyl-piperidinyl or alkyl-piperazinyl groups at position 10 modulated PTZ activity to a limited degree. Production of PTZ+* radicals was demonstrated by optical and ESR spectroscopy methods. PTZ+* radicals stability depended on their structure as demonstrated by promazine and thioridazine radicals. Thiol compounds such as GSH and N-acetylcysteine, L-tyrosine, L-tryptophan, the corresponding peptides, ascorbate and Trolox, prevented LADH inactivation by the MPO/H2O2/thioridazine system, in close agreement with their action as PTZ+* scavengers. NADH (not NAD+) produced transient protection of LADH against thioridazine and promazine radicals, the protection kinetics being affected by the relatively fast rate of NADH oxidation by these radicals. The role of the observed effects of PTZ radicals for PTZ cytotoxicity is discussed.