Dual Action of Antimonial Drugs on Thiol Redox Metabolism in the Human Pathogen Leishmania donovani

Abstract

Despite extensive use of antimonial compounds in the treatment of leishmaniasis, their mode of action remains uncertain. Here we show that trivalent antimony (Sb III ) interferes with trypanothione metabolism in drug-sensitive Leishmania parasites by two inherently distinct mechanisms. First, Sb III decreases thiol buffering capacity by inducing rapid efflux of intracellular trypanothione and glutathione in approximately equimolar amounts. Second, Sb III inhibits trypanothione reductase in intact cells resulting in accumulation of the disulfide forms of trypanothione and glutathione. These two mechanisms combine to profoundly compromise the thiol redox potential in both amastigote and promastigote stages of the life cycle. Furthermore, we demonstrate that sodium stibogluconate, a pentavalent antimonial used clinically for the treatment for leishmaniasis, induces similar effects on thiol redox metabolism in axenically cultured amastigotes. These observations suggest ways in which current antimony therapies could be improved, overcoming the growing problem of antimony resistance.Leishmania parasites cause a wide spectrum of human and animal infections ranging from life-threatening visceral disease to disfiguring mucosal and cutaneous forms of the disease. These "neglected" diseases are a significant cause of morbidity and mortality in 88 countries in the Americas, Africa, Asia, and Southern Europe; millions of people are at risk, and 400,000 new cases are reported annually (1). Leishmania spp. are obligate intracellular parasites of the vertebrate reticuloendothelial system, where they multiply as amastigotes in macrophage phagolysosomes; transmission is by blood-sucking sandflies, in which they proliferate as extracellular promastigotes. Treatment of the leishmaniases is far from ideal, and pentavalent antimonial (Sb V ) 1 preparations such as sodium stibogluconate (Pentostam) have been the front-line drugs for more than half a century. However, the clinical value of antimony therapy is now threatened because of the emergence of drug resistance (2) and co-infection with human immunodeficiency virus (3). Sb V is generally regarded as a pro-drug that first has to be activated by conversion to the trivalent form (Sb III ) (4). However, the site of reduction (host macrophage, amastigote, or both) and the mechanism of reduction (enzymatic or nonenzymatic) remain unclear (4 -8). The mode of action of these drugs is poorly understood. Sb III reversibly inhibits trypanothione reductase in vitro (9), but this has not been demonstrated in the intact parasite. However, inhibition of this unique and essential enzyme (10 -12) is of particular interest, because trypanothione (N 1 ,N 8Ϫ bis(glutathionyl) spermidine (T[SH] 2 )) is a key intermediate in the regulation of thiol redox homeostasis, as well as in defense against chemical (13,14) and oxidative stress (15, 16).Paradoxically, more is known about the mechanism of resistance to Sb III than its mode of action (17). A considerable body of evidence implicates trypanothione a...

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania

“…Although the mode of SbIII's action against Leishmania is poorly understood, mechanisms of resistance have been extensively described and involve the modulation of SbIII detoxifying pathways (36,37), altered uptake (19,38), increased efflux (39), and intracellular sequestration mediated by the ABC-thiol transporter multidrug resistance protein A (MRPA) (37,(40)(41)(42). The Cos-Seq screen for SbIII revealed 11 enriched cosmids ( Fig.…”

Expresión diferencial de proteínas en Leishmania (Viannia) panamensis asociadas con mecanismos de resistencia a antimoniato de meglumina

“…La cepa fue seleccionada hasta 37 mg/ml de antimonio pentavalente, debido a que se buscó tener un espectro de resistencia bastante amplio entre la cepa sensible y la resistente (CE 50 de 4 mg/ ml versus 37 mg/ml) y con mayor probabilidad de detectar proteínas asociadas al fenotipo resistente. En esta cepa resistente, se encontró expresión exagerada de las proteínas Hsp60 mitocondrial, Hsp70 mitocondrial y citosólica, proteína de isomerasa de disulfuro y dos proteínas hipotéticas nombradas como Sp(2) y Sp (25), las cuales aparentemente podrían estar participando en una respuesta inicial del parásito para evadir la acción leishmanicida del antimonio pentavalente de manera indirecta, contribuyendo al mecanismo primario de desintoxicación mediado por las moléculas de tripanotión-glutatión al eliminar las formas disulfuro de estos compuestos (19). Además, estas proteínas podrían contribuir a evitar la pérdida del potencial de membrana mitocondrial, controlar el potencial de oxidorreducción de la célula y evitar los procesos de apoptosis de fragmentación del ADN inducido por los antimoniales pentavalentes.…”

“…El antimonio pentavalente es suministrado como un promedicamento que requiere ser activado a antimonio trivalente; no se tiene claro si su activación se produce por una vía enzimática o de manera espontánea (19). Se han reportado varios blancos celulares para el antimonio pentavalente y el antimonio trivalente , incluyendo el tripanotión, que es el principal agente reductor de tioles en el parásito (20)(21)(22).…”

Assessing the Role of Potential Biomarkers in Antimony Susceptible and Resistant Clinical Isolates of L. donovani from India

“…The route of entry of SbV into Leishmania cells is still unknown but SbIII has been reported to be transported in Leishmania through aquaglyceroporin (AQP1) [11,12]. Recent evidence has suggested that part of the mode of ©NJB, Biotechnology Society of Nepal 2 Nepjol.info/index.php/njb action of SbV could be in depleting the cells of its reduced thiols [13]. Trypanothione (TSH), a major reduced thiol of Leishmania is a N 1 , N8 bisglutathione spermidine conjugate.…”