RESUMO
Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the Leishmania genus. Treatments available have limited safety and efficacy, high costs, and difficult administration. Thus, there is an urgent need for safer and more-effective therapies. Most trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. In previous studies, we showed that Leishmania amazonensis is highly susceptible to aryl-quinuclidines, such as E5700, which inhibit squalene synthase, and to the azoles itraconazole (ITZ) and posaconazole (POSA), which inhibit C-14α-demethylase. Herein, we investigated the antiproliferative, ultrastructural, and biochemical effects of combinations of E5700 with ITZ and POSA against L. amazonensis. Potent synergistic antiproliferative effects were observed against promastigotes, with fractional inhibitory concentration (FIC) ratios of 0.0525 and 0.0162 for combinations of E5700 plus ITZ and of E5700 plus POSA, respectively. Against intracellular amastigotes, FIC values were 0.175 and 0.1125 for combinations of E5700 plus ITZ and E5700 plus POSA, respectively. Marked alterations of the ultrastructure of promastigotes treated with the combinations were observed, in particular mitochondrial swelling, which was consistent with a reduction of the mitochondrial transmembrane potential, and an increase in the production of reactive oxygen species. We also observed the presence of vacuoles similar to autophagosomes in close association with mitochondria and an increase in the number of lipid bodies. Both growth arrest and ultrastructural/biochemical alterations were strictly associated with the depletion of the 14-desmethyl endogenous sterol pool. These results suggest the possibility of a novel combination therapy for the treatment of leishmaniasis.
Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Ergosterol/antagonistas & inibidores , Itraconazol/farmacologia , Leishmania mexicana/efeitos dos fármacos , Piridinas/farmacologia , Quinuclidinas/farmacologia , Triazóis/farmacologia , Tripanossomicidas/farmacologia , Animais , Meios de Cultura/química , Sinergismo Farmacológico , Quimioterapia Combinada , Ergosterol/biossíntese , Farnesil-Difosfato Farnesiltransferase/antagonistas & inibidores , Farnesil-Difosfato Farnesiltransferase/metabolismo , Humanos , Leishmania mexicana/isolamento & purificação , Leishmania mexicana/metabolismo , Leishmania mexicana/ultraestrutura , Leishmaniose Tegumentar Difusa/parasitologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/ultraestrutura , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Testes de Sensibilidade Parasitária , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/metabolismo , Esterol 14-Desmetilase/metabolismoRESUMO
A new Ca(2+) intracellular store, the acidocalcisome, has been reported in trypanosomatids. It has been characterized physiologically as a Ca(2+) store sensitive to nigericin. The Ca(2+)/H(+)-ATPase is the system responsible for Ca(2+) accumulation, which depends on a pH gradient formed by ATP- and PPi-dependent proton pumps. In this work we present physiological and morphological evidences for the presence of acidocalcisomes in Trypanosoma evansi. The parasites were purified and loaded with the fluorescent dye Fura 2-AM in order to detect the intracellular changes of Ca(2+) levels in individual cells. The simultaneous incubation of T. evansi cells with ionomycin and nigericin led to large release of Ca(2+) (ca. 200 nM) from intracellular stores, which was not observed with either agent alone. On the other hand, no enhancement of the nigericin-induced Ca(2+) release was observed in the presence of oligomycin. Additionally, the pretreatment with bafilomycin decreases the nigericin-induced Ca(2+) release. These results confirm the presence of an intracellular non-mitochondrial acidic Ca(2+) storage compartment. These results suggest that H(+)-ATPase is involved in the process of Ca(2+) accumulation into the acidocalcisomes. Furthermore, the cells loaded with acridine orange exhibited abundant fluorescent vacuoles, which were sensitive to nigericin or bafilomycin A(1). Electronic transmission microscopy observations demonstrated the presence of electron dense particles in the parasites. High levels of inorganic pyrophosphate and triphosphate were detected in perchloric acid extracts of T. evansi by high resolution 31P NMR. Taken together, these results present the first evidence for the presence of acidocalcisomes in T. evansi.
Assuntos
Cálcio/análise , Vesículas Citoplasmáticas/química , Macrolídeos , Trypanosoma/química , Trypanosoma/ultraestrutura , Laranja de Acridina , Animais , Antibacterianos/antagonistas & inibidores , Cálcio/metabolismo , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Sinergismo Farmacológico , Fluorescência , Ionomicina/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Masculino , Microscopia Eletrônica/métodos , Nigericina/metabolismo , Isótopos de Fósforo , Polifosfatos/análise , Polifosfatos/química , Ratos , Ratos Sprague-Dawley , Trypanosoma/metabolismoRESUMO
Molecular structural parameters of two potential drugs against Trypanosoma cruzi epimastigotes, 20-piperidin-2-yl-5alpha-pregnan-3beta,20-diol (1) and 20-N-methylpiperidin-2-yl-5alpha-pregnan-3beta, 20-diol (2) were studied using a combination of a stereoselective synthetic route, spectroscopic characterization and single-crystal X-ray analysis. Both compounds were synthesized with an R configuration at C20. This chirality is a consequence of the stereoselectivity observed during the formation of the intermediate 20-pyridin-2-yl-5alpha-pregnan-3beta,20R-diol (4). NMR data indicated that the six-membered aza ring of (2) is conformationally more restrained, in CDCl3 solution, than (1). X-ray studies showed that maximum deviations among structural molecular parameters of (1) and (2) correspond to torsion angles along the C20-C22 bonds, leading to a different relative orientation of the N atom; a critical structural parameter for the binding properties of aza-sterols to Delta(24(25)) sterol methyl transferase. Cremer-Pople parameters of the five-membered rings of (1) and (2) lie in the observed range for a family of tetracyclic fused ring systems retrieved from the CSD. The phi2 parameter of (1) lies just on the mean of the family, while phi2 of (2) deviates significantly towards the lower limit.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , Pregnanodiol/química , Pregnanodiol/farmacologia , Pregnanodiol/fisiologia , Trypanosoma cruzi/enzimologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Pregnanodiol/análogos & derivados , Pregnanodiol/síntese química , Estereoisomerismo , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
We investigated the mechanism of action of metabolically stable lysophospholipid analogues (LPAs), with potent anti-tumour and anti-protozoal activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Against the axenically grown epimastigote form of the parasite, the IC(50)s after 120 h for ET-18-OCH(3), miltefosine and ilmofosine were 3, 1 and 3 microM, respectively; at higher concentrations immediate lytic effects were observed. Eradication of the intracellular amastigote, grown inside Vero cells, was achieved at 0.1, 0.1 and 1 microM for ET-18-OCH(3), miltefosine and ilmofosine, respectively. Analysis of the lipid composition of epimastigotes exposed to LPAs at their IC(50) for 120 h showed that the ratio of phosphatidyl-choline (PC) to phosphatidylethanolamine (PE) changed from 1.5 in control cells to c. 0.67 in those treated with the analogues. A significant increase in the content of phosphatidylserine was also observed in treated cells. Intact epimastigotes efficiently incorporated radioactivity from L-[methyl-(14)C]methionine into PC, but not from [methyl-(14)C]choline. ET-18-OCH(3) inhibited the incorporation of L-[methyl-(14)C]methionine into PC with an IC(50) of 2 microM, suggesting that inhibition of the de novo synthesis through the Greenberg's pathway was a primary effect underlying the selective anti-parasitic activity of this compound. Antiproliferative synergism was observed as a consequence of combined treatment of epimastigotes with ET-18-OCH(3) and ketoconazole, a sterol biosynthesis inhibitor, probably due to the fact that a secondary effect of the latter is also a blockade of PC synthesis at the level of PE-PC-N-methyl-transferase.
Assuntos
Antiprotozoários/farmacologia , Cetoconazol/farmacologia , Lisofosfolipídeos/farmacologia , Esteróis/antagonistas & inibidores , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antifúngicos/farmacologia , Divisão Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Parasitária , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Esteróis/biossíntese , Esteróis/química , Trypanosoma cruzi/metabolismoRESUMO
The complexes [Cu(CTZ)(4)]Cl(2).2H(2)O (1), [Cu(CTZ)Cl(2)](2) (2), [Cu(KTZ)(3)Cl(2)] (3), and [Cu(KTZ)Cl(2)](2).2H(2)O (4) were prepared by reaction of CuCl(2) with CTZ and KTZ (where CTZ = 1-[[(2-chlorophenyl)diphenyl]methyl]-1H-imidazole and KTZ = cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine), respectively, in acetonitrile at different ligand to metal molar ratios. Gold complexes [Au(PPh(3))(CTZ)]PF(6) (5) and [Au(PPh(3))(KTZ)]PF(6).H(2)O (6) were synthesized by reaction of AuClPPh(3), with KPF(6) and CTZ or KTZ in acetonitrile. All the new compounds were characterized by NMR spectroscopy and microanalytical methods, and for the paramagnetic species EPR spectroscopy and DC magnetic susceptibility measurements were also employed. The solid-state structure of 1 has been determined by X-ray crystallography. 1 crystallizes in the triclinic space group P(-)1, with a = 12.773(2) A, b = 15.326(4) A, c = 11.641(2) A, V = 1957.4(7) A(3), Z = 1, and D(calcd) = 1.284 g/cm(3). The structure refinement converged at R1 = 0.0731 and wR2 = 0.1962. Complex 1 displayed a square-planar structure typical for tetrakis(imidazole)copper(II) complexes. The new compounds were tested for in vitro activity against cultures of epimastigotes of Trypanosoma cruzi, the causative agent of Chagas disease. At concentrations equivalent to 10(-6) M of total CTZ or KTZ (in DMSO) all the complexes exhibited significantly higher growth inhibitory activity than their respective parental compounds.
Assuntos
Antiprotozoários/química , Clotrimazol/química , Cobre/química , Ouro/química , Cetoconazol/química , Compostos Organometálicos/síntese química , Trypanosoma cruzi/efeitos dos fármacos , Algoritmos , Animais , Antiprotozoários/farmacologia , Doença de Chagas/patologia , Fenômenos Químicos , Físico-Química , Clotrimazol/farmacologia , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Cetoconazol/farmacologia , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Compostos Organometálicos/química , Compostos Organometálicos/farmacologiaRESUMO
The current situation regarding specific chemotherapy for Chagas disease (American trypanosomiasis), and new developments in this field, are reviewed. Despite previous controversy on the autoimmune origin of Chagas disease pathology, available knowledge supports the notion that this condition should be treated as a parasitic, not an autoimmune, disease. Currently available drugs (nitrofurans and nitroimidazoles) are active in acute or short-term chronic infections, but have very low antiparasitic activity against the prevalent chronic form of the disease, and toxic side-effects are frequently encountered. The nitroimidazole benznidazole has also shown significant activity in the treatment of reactivated Trypanosoma cruzi infections in patients with acquired immune deficiency syndrome and in other immunosuppressed patients with underlying chronic Chagas disease. Although the etiological agent, T. (Schizotrypanum) cruzi, requires specific endogenous sterols for cell viability and proliferation, the currently available antifungal sterol biosynthesis inhibitors are not powerful enough to induce parasitological cures of human or experimental infections. However, new triazole antifungal compounds, which are potent inhibitors of the sterol C14alpha demethylase of the parasite and have special pharmacokinetic properties, are capable of inducing parasitological cures in murine models of both acute and chronic Chagas disease. They are currently the most advanced candidates for clinical trials in patients with Chagas disease. Other potential chemotherapeutic agents against T. cruzi currently in development include antiproliferative lysophospholipid analogs (already in clinical trials as the first oral treatment for visceral leishmaniasis), cysteine proteinase (cruzipain) inhibitors, and compounds that interfere with purine salvage and inositol metabolism.
Assuntos
Doença de Chagas/tratamento farmacológico , Inibidores de Cisteína Proteinase/uso terapêutico , Humanos , Quinazolinas/uso terapêutico , Triazóis/uso terapêuticoRESUMO
We describe the in vitro antiproliferative effects of the new triazole derivative UR-9825 against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease in Latin America. The compound was found to be extremely active against the cultured (epimastigote) form of the parasite, equivalent to that present in the reduviid vector, with a MIC of 30 nM, a concentration 33-fold lower than that required with the reference compound ketoconazole. At that MIC, growth arrest coincided with depletion of the parasite's 4,14-desmethyl endogenous sterols (ergosterol, 24-ethylcholesta-5,7,22-trien-3b-ol, and precursors) and their replacement by methylated sterols (lanosterol, 24-methylenedihydrolanosterol, and obtusifoliol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of UR-9825 was inhibition of the parasite's sterol C14alpha demethylase, as seen with other azole derivatives. The phospholipid composition of growth-arrested epimastigotes was also altered, when compared to controls, with a significant increase in the content of phosphatidylethanolamine and phosphatidylserine and a concomitant reduction of the content of phosphatidylcholine. The clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37 degrees C, was even more sensitive to UR-9825, with a MIC of 10 nM, comparable to that for ketoconazole. The results showed that UR-9825 is among the most potent azole derivatives tested against this parasite and support in vivo studies with this compound.
Assuntos
Antiprotozoários/farmacologia , Quinazolinas/farmacologia , Triazóis/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Fosfolipídeos/química , Relação Estrutura-Atividade , Trypanosoma cruzi/citologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismoRESUMO
We report the in vivo activity of the bis-triazole derivative D0870 against a variety of strains of Trypanosoma cruzi, the causative agent of Chagas' disease, including nitroimidazole/nitrofuran-resistant strains. In both acute and chronic murine models of the disease, treatment with D0870 at < or = 10 mg/kg on alternate days for a total of 20 doses provided 60-100% protection against death, regardless of the drug sensitivity of the infecting strain. In the acute model we obtained 70-100% parasitological cure in seven of the nine strains tested, including one intermediate and two highly drug-resistant strains. D0870 was able to cure 30-45% of animals chronically infected with various strains, including those harbouring the Colombiana strain, among which no cures were obtained with benznidazole. We also found that the anti-T. cruzi activity of D0870 is largely retained even if the hosts are immunosuppressed.
Assuntos
Doença de Chagas/tratamento farmacológico , Triazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Doença de Chagas/parasitologia , Resistência a Medicamentos , Feminino , Camundongos , Triazóis/uso terapêuticoRESUMO
L-Carnitine plays an important role in the mitochondrial uptake of long-chain fatty acids in mammals. It has recently been shown that this compound has a marked hypo-cholesterolemic effect when used in conjunction with lipid-rich diets. The aim of this study was to investigate the effects of L-carnitine on the fatty acid composition of plasma lipoproteins in rabbits fed with different diets. Four different groups were investigated: group I (standard diet), group II (standard diet supplemented with L-carnitine at 80 mg/kg), group III (standard diet supplemented with 0.5% cholesterol), and group IV (standard diet supplemented with 0.5% cholesterol plus L-carnitine at 80 mg/kg). The feeding period was 126 d. Total plasma cholesterol was indistinguishable in groups I and II, but increased nearly 40-fold in group III. This increment was reduced by 50% in group IV. Correspondingly, total cholesterol content in lipoprotein fractions [very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) separated by agarose gel chromatography was the same for groups I and II, while for animals fed a cholesterol-rich diet (III) total cholesterol in VLDL + LDL increased nearly 100-fold when compared with groups I and II but, again, the increment was reduced by 50% in group IV. In contrast, total cholesterol in HDL increased only fivefold for both groups III and IV when compared with groups I and II, indicating no effects of L-carnitine on this parameter. The reduction of total cholesterol in VLDL + LDL particles in animals fed a cholesterol-rich diet plus L-carnitine was associated with a marked decrease in the ratio of cholesteryl ester to free cholesterol and a dramatic increase in their phospholipid content; opposite effects were observed for HDL. L-Carnitine induced a marked decrease in the saturated to unsaturated C16 + C18 fatty acid ratio in cholesteryl esters associated with VLDL and LDL from animals fed with both normal and cholesterol-rich diets. The opposite effect (a large increase in the saturated to unsaturated fatty acid ratio) was observed for both cholesteryl esters and phospholipids associated with HDL in animals fed with both diets. The results suggested that the hypocholesterolemic effects of L-carnitine could be associated with increased systemic breakdown of cholesteryl esters, a probable increase in reverse cholesterol transport, and the stabilization of a phospholipid-based structure of VLDL + LDL particles.
Assuntos
Carnitina/farmacologia , Colesterol na Dieta/administração & dosagem , Lipoproteínas/sangue , Animais , Ésteres do Colesterol/sangue , Ésteres do Colesterol/química , Ácidos Graxos/sangue , Lipoproteínas/classificação , Masculino , CoelhosRESUMO
We have studied the in vivo activity of the new experimental triazole derivative SCH 56592 (posaconazole) against a variety of strains of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, in both immunocompetent and immunosuppressed murine hosts. The T. cruzi strains used in the study were previously characterized as susceptible (CL), partially resistant (Y), or highly resistant (Colombiana, SC-28, and VL-10) to the drugs currently in clinical use, nifurtimox and benznidazole. Furthermore, all strains are completely resistant to conventional antifungal azoles, such as ketoconazole. In the first study, acute infections with the CL, Y, and Colombiana strains in both normal and cyclophosphamide-immunosuppressed mice were treated orally, starting 4 days postinfection (p.i.), for 20 consecutive daily doses. The results indicated that in immunocompetent animals SCH 56592 at 20 mg/kg of body weight/day provided protection (80 to 90%) against death caused by all strains, a level comparable or superior to that provided by the optimal dose of benznidazole (100 mg/kg/day). Evaluation of parasitological cure revealed that SCH 56592 was able to cure 90 to 100% of the surviving animals infected with the CL and Y strains and 50% of those which received the benznidazole- and nifurtimox-resistant Colombiana strain. Immunosuppression markedly reduced the mean survival time of untreated mice infected with any of the strains, but this was not observed for the groups which received SCH 56592 at 20 mg/kg/day or benznidazole at 100 mg/kg/day. However, the overall cure rates were higher for animals treated with SCH 56592 than among those treated with benznidazole. The results were confirmed in a second study, using the same model but a longer (43-dose) treatment period. Finally, a model for the chronic disease in which oral treatment was started 120 days p.i. and consisted of 20 daily consecutive doses was investigated. The results showed that SCH 56592 at 20 mg/kg/day was able to induce a statistically significant increase in survival of animals infected with all strains, while benznidazole at 100 mg/kg/day was able to increase survival only in animals infected with the Colombiana strain. Moreover, the triazole was able to induce parasitological cures in 50 to 60% of surviving animals, irrespective of the infecting strain, while no cures were obtained with benznidazole. Taken together, the results demonstrate that SCH 56592 has in vivo trypanocidal activity, even against T. cruzi strains naturally resistant to nitrofurans, nitroimidazoles, and conventional antifungal azoles, and that this activity is retained to a large extent in immunosuppressed hosts.
Assuntos
Triazóis/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Doença Aguda , Animais , Doença de Chagas/tratamento farmacológico , Doença Crônica , Resistência a Medicamentos , Feminino , Imunocompetência , Terapia de Imunossupressão , Camundongos , Nitroimidazóis/farmacologiaRESUMO
A detailed analysis of the endogenous sterols present in the clinically relevant intracellular (amastigote) stages of Trypanosoma cruzi, is presented. The parasites were grown in cultured Vero cells in the absence or presence of different sterol biosynthesis inhibitors, including the C14alpha demethylase inhibitor ketoconazole and two inhibitors of delta24(25)-sterol methyl transferase, 20 piperidin-2-yl-5alpha-pregnan-3beta-20-R-diol (22,26-azasterol) and 24-(R,S),25-epiminolanosterol. Amastigotes were isolated and purified from their host cells and neutral lipids were extracted, separated and analyzed by chromatographic and mass spectrometric methods. Control (untreated) amastigotes contained as main endogenous sterols 24-methyl-cholesta-7-en-3beta-ol (ergosta-7-en-3beta-ol) and its 24-ethyl analog, plus smaller amounts of their precursor, ergosta-7,24(28)dien-3beta-ol; these cells also contained cholesterol (up to 80% by weight of total sterols), probably derived from host cells. Amastigotes that proliferated in the presence of 10 nM ketoconazole (minimal inhibitory concentration, MIC) for 24 h had a sharply reduced content of endogenous 4-desmethyl sterols with a concomitant accumulation of 24-methyl-dihydrolanosterol and 24-methylene-dihydrolanosterol. On the other hand, amastigotes incubated during the same period of time with the two inhibitors of 24(25)-SMT at their respective MICs (100-300 nM) accumulated large amounts of C27 sterols whose structure suggested, in the case of 22,26-azasterol, that delta14 sterol reductase was also inhibited. Ketoconazole produced a dose-dependent reduction in the incorporation of [2-(14)C]-acetate into the parasite's endogenous C4-desmethyl sterols with an IC50 of 50 nM, indistinguishable from the value reported previously for the extracellular epimastigote form. Taken together, the results showed that amastigotes have a simpler sterol biosynthetic pathway than that previously described for epimastigotes, lacking both delta5 and delta22 reductases. They also suggest that the 100-fold higher potency of antifungal azoles as antiproliferative agents against amastigotes, when compared with epimastigotes, is most probably due to a smaller pool of endogenous sterols in the intracellular parasites.
Assuntos
Esteróis/biossíntese , Trypanosoma cruzi/metabolismo , Animais , Chlorocebus aethiops , Colestanol/análogos & derivados , Colestanol/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Cetoconazol/farmacologia , Lanosterol/análogos & derivados , Lanosterol/farmacologia , Metiltransferases/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Esterol 14-Desmetilase , Trypanosoma cruzi/química , Trypanosoma cruzi/efeitos dos fármacos , Células VeroRESUMO
Current developments in experimental chemotherapy of Chagas' disease are reviewed, in particular the demonstration that fourth-generation azole derivatives (inhibitors of sterol C14alpha demethylase), with particular selectivity against Trypanosoma cruzi and special pharmacokinetic properties, are capable of inducing radical parasitological cures in murine models of both acute and chronic disease. These are the first reports of parasitological cure of this disease in its chronic phase. We also discuss the relevance of etiological treatment in the clinical outcome of patients with chronic Chagas' disease. Although previous studies have suggested an important autoimmune component in the pathogenesis of this disease, recent results obtained using highly sensitive polymerase chain reaction based detection methods and detailed immunological characterization of the inflammatory process associated with chagasic cardiomyopathy indicate a positive correlation between tissue parasitism and the severity of cardiac pathological findings. Effective antiparasitic treatment can lead to regression of the inflammatory heart lesions and fibrosis in experimental animals and to stop the progression of the disease in humans. Taken together, these findings support the notion that the presence of the parasite is a necessary and sufficient condition for chagasic cardiomyopathy and confirm the importance of specific etiological treatment in the management of chronic chagasic patients.
Assuntos
Doença de Chagas/tratamento farmacológico , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Animais , Doença de Chagas/imunologia , Humanos , Modelos Químicos , Esteróis/antagonistas & inibidores , Esteróis/biossíntese , Triazóis/uso terapêuticoRESUMO
We have investigated the antiproliferative effects of SCH 56592, a new experimental triazole, against Trypanosoma (Schizotrypanum) cruzi, the etiological agent of Chagas' disease in Latin America. SCH 56592 blocked the proliferation of the epimastigote form of the parasite in vitro at 30 nM, a concentration 30- to 100-fold lower than that required with the reference compounds ketoconazole and itraconazole. At that concentration all the parasite's endogenous sterols (ergosterol, 24-ethyl-cholesta-5,7,22-trien-3 beta-ol, and its 22-dihydro analogs), were replaced by methylated sterols (lanosterol and 24-methylene-dihydrolanosterol), as revealed by high-resolution gas chromatography coupled with mass spectrometry. This indicated that the primary mechanism of action of the drug was inhibition of the parasite's sterol C-14 alpha demethylase. Against the clinically relevant intracellular amastigote form, grown in cultured Vero cells at 37 degrees C, the MIC of SCH 56592 was 0.3 nM, again 33- to 100-fold lower than that of ketoconazole or itraconazole. In a murine model of acute Chagas' disease, SCH 56592 given at > or = 10 mg/kg of body weight/day for a total of 43 doses allowed 85 to 100% survival and 90 to 100% cure of the surviving animals, as verified by parasitological, serological, and PCR-based tests, while ketoconazole given at 30 mg/kg day allowed 60% survival but only 20% cure. In a murine model of chronic Chagas' disease, SCH 56592 was again more effective than ketoconazole, providing 75 to 85% protection from death, with 60 to 75% parasitological cures of the surviving animals, while no parasitological cures were observed with ketoconazole. The results indicate that SCH 56592 is the most powerful sterol biosynthesis inhibitor ever tested against T. cruzi and may be useful in the treatment of human Chagas' disease.
Assuntos
Antiparasitários/farmacologia , Doença de Chagas/tratamento farmacológico , Triazóis/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Camundongos , Triazóis/uso terapêutico , TripanossomicidasRESUMO
The subcellular localization of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, which catalyzes the first committed step of the mevalonate pathway, was investigated in Trypanosoma cruzi epimastigotes using well-established cell fractionation procedures. It was found that ca. 80% of the activity of the enzyme was associated with the glycosomes, microbody-like organelles unique to kinetoplastid protozoa which contain most of the enzymes of the glycolytic pathway, while the rest of the activity was found in the soluble (cytoplasmatic) fraction, with almost no activity associated with microsomes. The glycosome-associated enzyme is not membrane-bound as it was recovered quantitatively in the aqueous phase of the biphasic system formed by Triton X-114 at 30 degrees C. Studies with digitonin-permeabilized intact epimastigotes demonstrated the presence of two pools of soluble HMG-CoA reductase in these cells, associated to the cytoplasmic and glycosomal compartments. Steady-state kinetic studies of the glycosome-associated enzyme indicated classical Michaelis-Menten behavior with Km,app (HMG-CoA) 28 +/- 3 microM, Km,app (NADPH) 37 +/- 4 microM, and Vm,app 3.9 +/- 0.2 nmol/min mg protein; the transition-state analog lovastatin behaved as a competitive inhibitor with respect to HMG-CoA with Kis 23 nM and a noncompetitive inhibitor toward NADPH with Kii 29 nM. The results are in complete agreement with recent gene cloning and expression studies which showed that T. cruzi HMG-CoA reductase lacks the NH2-terminal membrane-spanning sequence. This is the first demonstration of a soluble eukaryotic HMG-CoA reductase and also the first report on the presence of an enzyme of the isoprenoid biosynthesis pathway in glycosomes.
Assuntos
Hidroximetilglutaril-CoA Redutases/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Fracionamento Celular , Digitonina , Glicólise , Hidroximetilglutaril-CoA Redutases/isolamento & purificação , Cinética , Organelas/enzimologia , Solubilidade , Frações Subcelulares/enzimologia , Trypanosoma cruzi/metabolismoRESUMO
Detailed analysis of the endogenous sterol content of purified Pneumocystis carinii preparations by gas-liquid chromatography coupled to mass spectrometry suggested that this parasite can both synthesize de novo steroid skeletons (to produce delta7 sterols) and take them from the infected host (leading to delta5 sterols). In both cases the final products are 24-alkyl sterols, resulting from the action of delta24(25) and delta24(24') sterol methyltransferases, enzymes not present in vertebrates. To investigate the physiological significance of these sterols, cultures of P. carinii in embryonic lung cells were exposed to 22,26-azasterol (20-piperidin-2-yl-5alpha-pregnan-3beta-20(R)-diol), a compound previously shown to inhibit both enzymes and to halt cell proliferation in fungi and protozoa. This compound produced a dose-dependent reduction in the parasite proliferation, with a 50% inhibitory concentration of 0.3 microM and 80% reduction of growth after 96 h at 10 microM. Correspondingly, parasites treated with the azasterol at 10 microM for 48 h accumulated 24-desalkyl sterols such as zymosterol (cholesta-8,24-dien-3beta-ol) and cholesta-8,14,24-trien-3beta-ol to ca. 40% of the total mass of endogenous sterols. This is the first report on the antiproliferative effects of a sterol biosynthesis inhibitor on P. carinii and indicate that sterol methyltransferase inhibitors could be the basis of a novel and specific chemotherapeutic approach to the treatment of P. carinii infections.
Assuntos
Inibidores Enzimáticos/farmacologia , Metiltransferases/antagonistas & inibidores , Pneumocystis/efeitos dos fármacos , Esteróis/biossíntese , Alquilação , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Colestanol/análogos & derivados , Colestanol/farmacologia , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pneumocystis/metabolismoRESUMO
The accepted mechanism for the antiproliferative effects of sterol biosynthesis inhibitors (SBI) against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi, the causative agent of Chagas' disease, is the depletion of specific parasite sterols that are essential growth factors and cannot be replaced by cholesterol, the main sterol present in the vertebrate host. However, the precise metabolic roles of these specific parasite sterols are unknown. We approached this problem by subjecting T. cruzi epimastigotes to two types of SBI, inhibitors of sterol C-14 demethylase and delta 24(25) methyl transferase, and investigating the modification of lipid composition and enzyme activities in the plasma membranes of the parasite. We found in purified plasma membrane from SBI-treated cells that, together with the expected changes in the sterol composition, there was also an inversion of the phosphatidylcholine (PC) to phosphatidylethanolamine (PE) ratio and a large increase in the content of saturated fatty acids esterified to phospholipids. The modification of the phospholipid headgroup composition correlated with a 70% reduction in the specific activity of the membrane-bound PC-PE-N-methyl transferase SBI-treated cells; it was shown that this inhibition was not due to a direct effect of the drug on the enzyme. Finally, the specific activity of the Mg(2+)-dependent, vanadate-sensitive ATPase present in the membranes was also inhibited by ca. 50% in SBI-treated cells. The results suggest that one of the primary effects of the depletion of endogenous sterols induced by SBI in T. cruzi is a modification of the cellular phospholipid composition as a consequence of a reduced activity of PE-PC-N-methyl transferase and probably of the acyl delta 9 and delta 6 desaturases; this, in turn, could affect the activity of other enzymatic and transport proteins.
Assuntos
Colestanol/análogos & derivados , Cetoconazol/farmacologia , Lanosterol/análogos & derivados , Lipídeos de Membrana/análise , Fosfolipídeos/análise , Esteróis/biossíntese , Trypanosoma cruzi/efeitos dos fármacos , Animais , ATPase de Ca(2+) e Mg(2+)/efeitos dos fármacos , Colestanol/farmacologia , Lanosterol/farmacologia , Esteróis/antagonistas & inibidores , Trypanosoma cruzi/químicaRESUMO
We report the ultrastructural alterations induced on the proliferative stages of Trypanosoma cruzi, the causative agent of Chagas' disease, by two Delta(24(25)) sterol methyl transferase (24(25)-SMT) inhibitors, 22,26-azasterol and 24(R,S),25-epiminolanosterol. Both compounds are sterol biosynthesis inhibitors which had previously been shown to be potent growth inhibitors and whose effects are potentiated by the C14a demethylase inhibitor, ketoconazole. Epimastigotes treated with the minimal growth inhibitory concentration of 22,26-azasterol (10 microM) for 144 h, which were completely depleted of endogenous 4-desmethyl sterols and accumulated 24-desalkyl sterols, showed the appearance of electron-dense granules, mitochondrial swelling and intense vacuolization. At high concentration (> or = 30 microM) the sterol analog induced gross alterations in the organization of chromatin and rapid cell lysis. The treatment of epimastigotes with 24-(R,S),25-epiminolanosterol induced at low concentrations (1 microM) alterations similar to those observed with 22,26-azasterol but additionally modifications of the kinetoplast were observed. Higher concentrations (> or = 3 microM) induced total lysis. The combination of both sterol analogs with ketoconazole, at sub-optimal concentrations, induced the same alterations as 22,26-azasterol (10 microM) or epiminolanosterol (1 microM). The results confirm the conclusions of previous studies which indicated that one important cytotoxic effects of sterol biosynthesis inhibitors in this organism is the alteration of the parasite's mitochondrial system.
RESUMO
Inhibitors of sterol and phospholipid biosynthesis in kinetoplastid parasites such as Trypanosoma cruzi, the causative agent of Chagas' disease, and different species of Leishmania have potent and selective activity as chemotherapeutic agents in vitro and in vivo. Recent work with the sterol C14 alpha-demethylase inhibitor D0870, a bis triazole derivative, showed that this compound is capable of inducing radical parasitological cure in murine models of both acute and chronic Chagas' disease. Other inhibitors of this type, such as SCH 56592, have also shown curative, rather than suppressive, activity against T. cruzi in these models. Leishmania species have different susceptibilities to sterol biosynthesis inhibitors, both in vitro and in vivo. Leishmania braziliensis promastigotes, naturally resistant to C14 alpha-demethylase inhibitors such as ketoconazole and D0870, were susceptible to these drugs when used in combination with the squalene epoxidase inhibitor terbinafine. Inhibitors of delta 24(25) sterol methyl transferase have been shown to act as potent antiproliferative agents against Trypanosoma cruzi, both in vitro and in vivo. New inhibitors of this type which show enhanced activity and novel mechanisms of action have been synthesized. Recent work has also demonstrated that this type of enzyme inhibitors can block sterol biosynthesis and cell proliferation in Pneumocystis carinii, a fungal pathogen which had previously been found resistant to other sterol biosynthesis inhibitors. Ajoene, an antiplatelet compound derived from garlic, was shown to have potent antiproliferative activity against epimastigotes and amastigotes of Trypanosoma cruzi in vitro; this activity was associated with a significant alteration of the phospholipid composition of the cells with no significant effects on the sterol content. In addition, alkyllsophospholipids such as ilmofosine, miltefosine and edelfosine have been shown to block the proliferation of T. cruzi and Leishmania and alter both the phospholipid and sterol composition. These results indicate the potential of lipid biosynthesis inhibitors as useful therapeutic agents in the treatment of leishmaniasis and Chagas' disease.