The Mitochondrion Is a Site of Trypanocidal Action of the Aromatic Diamidine DB75 in Bloodstream Forms of Trypanosoma brucei

Abstract

Human African trypanosomiasis (HAT) is a fatal tropical disease caused by infection with protozoans of the speciesNew therapies are desperately needed for the treatment of human African trypanosomiasis (HAT). This reemerging tropical disease afflicts as many as 500,000 people (2, 5). Current drugs, such as pentamidine, suramin, melarsoprol, eflornithine, and nifurtimox, are often associated with deleterious side effects and cannot be administered orally (5,16,19,34).A promising new therapy, DB289 [2,5-bis-(4-amidinophenyl)-furan bis-O-methylamidoxime], is undergoing phase III clinical trials for registration as the first orally administered drug for early-stage HAT (19). The oral prodrug DB289 is metabolically converted to the trypanocidal agent DB75 [2,5-bis(4-amidinophenyl)furan] (6, 50, 51). DB75 is active against Trypanosoma spp. in vitro (11,12) and is a structural analogue of the aromatic diamidine drug pentamidine.DB75 and pentamidine are antimicrobial diamidine-type compounds that are active against various fungal and protozoal infections. However, the mechanism through which diamidine drugs exert their activity is still not entirely known and continues to be a focus of extensive research (46). We previously investigated the mechanisms of action of DB75 and pentamidine in yeast cells and found that both drugs appear to disrupt the mitochondrial function in Saccharomyces cerevisiae by collapsing the mitochondrial membrane potential (⌿ m ) and inhibiting mitochondrial respiration (24). We also showed that DB75 and pentamidine inhibit oxidative phosphorylation in isolated rat liver mitochondria (24), which is consistent with the results attained with pentamidine in a previous study (31).The purpose of this investigation was to gain insight into the mechanism of trypanocidal action of DB75. In particular, we investigated if DB75 acts against Trypanosoma brucei bloodstream forms (BFs) by targeting the mitochondrion. MATERIALS AND METHODSChemicals. DB75 [2,5-bis(4-amidinophenyl)furan dihydrochloride] was obtained from David Boykin at Georgia State University, Atlanta, and pentamidine isethionate [1,5-di(4-amidinophenoxy)pentane isethionate] was prepared by LyphoMed, Inc. (Melrose Park, IL). All other chemicals were of the highest quality available and were from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.Collection and isolation of trypanosomes from infected blood. Male SpragueDawley rats and Swiss-Webster mice (Charles River Laboratories, Cambridge, MA) were infected by intraperitoneal injection of approximately 2 ϫ 10 6 and 5 ϫ 10 5 BFs of Trypanosoma brucei brucei strain 427 cells, respectively. Infected blood was collected through cardiac puncture at the time of peak parasitemia (usually day 5 postinfection for rats and day 4 postinfection for mice). The blood was centrifuged in heparinized tubes at 2,200 ϫ g for 10 min at 4°C, and the buffy coat was removed and diluted 1:3 in phosphate saline glucose buffer, pH 8.0. The trypanosomes were purified from the blood by the use of DE52 anion-exchange...

Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes

“…These drugs selectively cleave kinetoplast DNA (25), and ethidium bromide also targets nuclear DNA (35,36). In addition, pentamidine disrupts ΔΨ mito (37), and this may be the basis of pentamidine sensitivity, demonstrated by us and by others (5), in T. brucei with dispensable kinetoplasts. Ultimately, ISM and diminazene aceturate resistance are of major concern (38), and some resistant field isolates appear to depend on adaptations that remain to be identified (6).…”

Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes

“…Melarsoprol has been suggested to act primarily by forming a toxic adduct with trypanothione, known as Mel T (5, 6), whereas pentamidine is a DNA-binding drug that becomes highly concentrated in trypanosomes and collapses the mitochondrial membrane potential (7). Arsenical/diamidine cross-resistance was first reported over 60 y ago (8).…”

Contributions of Ultrastructural Studies to the Cell Biology of Trypanosmatids: Targets for Anti-Parasitic Drugs

“…In T. cruzi, some of the new compounds (classified as reversed amidines) presented a high level of activity against amastigotes and trypomastigotes both in vivo and in vitro [18][19][20][21][22][23][24][25][26]. Ultrastructural analysis showed that the different aromatic diamines displayed similar effects in T. brucei, T cruzi and Leishmania, characterized by the presence of swollen mitochondria presenting low electron-density structures, fragmentation of the membrane and crista and mitochondrial disruption [12,13,18,20,23,24,[27][28][29][30][31][32].…”