Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

Abstract

Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH) was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1) was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect) forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase.

9-(2′-Deoxy-2′-Fluoro-β- d -Arabinofuranosyl) Adenine Is a Potent Antitrypanosomal Adenosine Analogue That Circumvents Transport-Related Drug Resistance

“…The cell cycle progression of bloodstream-form T. brucei (strain Lister 427) treated with FANA-A was assessed by visualizing the DNA in the nuclei and kinetoplasts by use of the fluorescent dye DAPI (4=,6-diamidino-2-phenylindole), followed by microscopic analysis as described previously (53). Briefly, 50 l of a trypanosome suspension (5 ϫ 10 5 cells/ml) was spread onto a glass microscope slide and was left to dry before methanol fixation overnight at 20°C.…”

Antitrypanosomal Activity of a Novel Taccalonolide from the Tubers of Tacca leontopetaloides

“…However, much work is needed in order to delineate the anti‐microbial potential of Tacca species as observed in their traditional usage. Therefore, we give an initial report of the anti‐trypanocidal potential of Tacca leontopetaloides , using the highly standardised model organism Trypansonsoma brucei brucei s427 lister strain (Alkhaldi et al ., ) for a first assessment of its antiprotozoal properties. Eight fractions were found to have various degrees of trypanocidal activity, relatively from very low activity (EC 50 > 20 µg/mL, result not shown), to moderate activity (EC 50 < 20 µg/mL), and high activity (EC 50 < 5 µg/mL), with the most interesting fraction being TL 80‐84 with an EC 50 of 0.79 ± 0.029 µg/mL (Table ).…”

Discovery of Sustainable Drugs for Neglected Tropical Diseases: Cashew Nut Shell Liquid (CNSL)‐Based Hybrids Target Mitochondrial Function and ATP Production in Trypanosoma brucei

“…The pellet was washed once in 1 mL of PBS (pH 7.4) and resuspended in 1 mL of PBS containing 200 n m TMRE; the cells then were incubated at 37 °C for 30 min and subsequently placed on ice for another 30 min before analysis on a Becton Dickinson FACSCalibur using a FL2‐height detector, and CellQuest and FlowJo software. Valinomycin (Sigma–Aldrich; 100 n m ) and troglitazone (Sigma–Aldrich; 10 μ m ) were used as controls for mitochondrial membrane depolarization and positive hyperpolarization, respectively …”