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.

Institute of Infection Immunity and Inflammation College of Medical Veterinary and Life Sciences University of Glasgow Glasgow United Kingdom

Institute of Parasitology Biology Centre and Faculty of Science University of South Bohemia České Budějovice Czech Republic

Instituto de Química Médica IQM CSIC Juan de la Cierva 3 E 28006 Madrid Spain

Zobrazit více v PubMed

Acestor N., Zikova A., Dalley R.A., Anupama A., Panigrahi A.K., Stuart K.D. Trypanosoma brucei mitochondrial respiratome: composition and organization in procyclic form. Mol. Cell Proteom. 2011;10 M110 006908. PubMed PMC

Allemann N., Schneider A. ATP production in isolated mitochondria of procyclic Trypanosoma brucei. Mol. Biochem. Parasitol. 2000;111:87–94. PubMed

Beck K., Acestor N., Schulfer A., Anupama A., Carnes J., Panigrahi A.K., Stuart K. Trypanosoma brucei Tb927.2.6100 is an essential protein associated with kinetoplast DNA. Eukaryot. Cell. 2013;12:970–978. PubMed PMC

Benein P., Almuteri M.A., Mehanna A.S., D'Souza G.G. Synthesis of triphenylphosphonium phospholipid conjugates for the preparation of mitochondriotropic liposomes. Methods Mol. Biol. 2015;1265:51–57. PubMed

Birch-Machin M.A., Turnbull D.M. Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues. Methods Cell Biol. 2001;65:97–117. PubMed

Bowler M.W., Montgomery M.G., Leslie A.G., Walker J.E. How azide inhibits ATP hydrolysis by the F-ATPases. Proc. Natl. Acad. Sci. U. S. A. 2006;103:8646–8649. PubMed PMC

Bruhn D.F., Sammartino M.P., Klingbeil M.M. Three mitochondrial DNA polymerases are essential for kinetoplast DNA replication and survival of bloodstream form Trypanosoma brucei. Eukaryot. Cell. 2011;10:734–743. PubMed PMC

Brun R., Blum J., Chappuis F., Burri C. Human African trypanosomiasis. Lancet. 2010;375:148–159. PubMed

Cairns A.G., McQuaker S.J., Murphy M.P., Hartley R.C. Targeting mitochondria with small molecules: the preparation of MitoB and MitoP as exomarkers of mitochondrial hydrogen peroxide. Methods Mol. Biol. 2015;1265:25–50. PubMed

Chaudhuri M., Ajayi W., Hill G.C. Biochemical and molecular properties of the Trypanosoma brucei alternative oxidase. Mol. Biochem. Parasitol. 1998;95:53–68. PubMed

Cortes L.A., Castro L., Pesce B., Maya J.D., Ferreira J., Castro-Castillo V., Parra E., Jara J.A., Lopez-Munoz R. Novel gallate triphenylphosphonium derivatives with potent antichagasic activity. PLoS One. 2015;10:e0136852. PubMed PMC

Coustou V., Biran M., Breton M., Guegan F., Riviere L., Plazolles N., Nolan D., Barrett M.P., Franconi J.M., Bringaud F. Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei. J. Biol. Chem. 2008;283:16342–16354. PubMed

Dardonville C., Alkhaldi A.A., De Koning H.P. SAR studies of diphenyl cationic trypanocides: superior activity of phosphonium over ammonium salts. ACS Med. Chem. Lett. 2015;6:151–155. PubMed PMC

Dardonville C., Barrett M.P., Brun R., Kaiser M., Tanious F., Wilson W.D. DNA binding affinity of bisguanidine and bis(2-aminoimidazoline) derivatives with in vivo antitrypanosomal activity. J. Med. Chem. 2006;49:3748–3752. PubMed

Delespaux V., de Koning H.P. Drugs and drug resistance in African trypanosomiasis. Drug Resist. Updates. 2007;10:30–50. PubMed

Denninger V., Figarella K., Schonfeld C., Brems S., Busold C., Lang F., Hoheisel J., Duszenko M. Troglitazone induces differentiation in Trypanosoma brucei. Exp. Cell Res. 2007;313:1805–1819. PubMed

Desquesnes M., Dargantes A., Lai D.H., Lun Z.R., Holzmuller P., Jittapalapong S. Trypanosoma evansi and surra: a review and perspectives on transmission, epidemiology and control, impact, and zoonotic aspects. Biomed. Res. Int. 2013:321237. PubMed PMC

Figarella K., Uzcategui N.L., Beck A., Schoenfeld C., Kubata B.K., Lang F., Duszenko M. Prostaglandin-induced programmed cell death in Trypanosoma brucei involves oxidative stress. Cell Death Differ. 2006;13:1802–1814. PubMed

Gnipova A., Panicucci B., Paris Z., Verner Z., Horvath A., Lukes J., Zikova A. Disparate phenotypic effects from the knockdown of various Trypanosoma brucei cytochrome c oxidase subunits. Mol. Biochem. Parasitol. 2012;184:90–98. PubMed

Gnipova A., Subrtova K., Panicucci B., Horvath A., Lukes J., Zikova A. The ADP/ATP carrier and its relationship to OXPHOS in an ancestral protist Trypanosoma brucei. Eukaryot. Cell. 2015;14:297–310. PubMed PMC

Gould M.K., Bachmaier S., Ali J.A., Alsford S., Tagoe D.N., Munday J.C., Schnaufer A.C., Horn D., Boshart M., de Koning H.P. Cyclic AMP effectors in African trypanosomes revealed by genome-scale RNA interference library screening for resistance to the phosphodiesterase inhibitor CpdA. Antimicrob. Agents Chemother. 2013;57:4882–4893. PubMed PMC

Gould M.K., Vu X.L., Seebeck T., de Koning H.P. Propidium iodide-based methods for monitoring drug action in the kinetoplastidae: comparison with the Alamar Blue assay. Anal. Biochem. 2008;382:87–93. PubMed

Guler J.L., Kriegova E., Smith T.K., Lukes J., Englund P.T. Mitochondrial fatty acid synthesis is required for normal mitochondrial morphology and function in Trypanosoma brucei. Mol. Microbiol. 2008;67:1125–1142. PubMed PMC

Hammarton T.C., Mottram J.C., Doerig C. The cell cycle of parasitic protozoa: potential for chemotherapeutic exploitation. Prog. Cell Cycle Res. 2003;5:91–101. PubMed

Hanson W.L., Chapman W.L., Jr., Kinnamon K.E. Testing of drugs for antileishmanial activity in golden hamsters infected with Leishmania donovani. Int. J. Parasitol. 1977;7:443–447. PubMed

Hiltensperger G., Jones N.G., Niedermeier S., Stich A., Kaiser M., Jung J., Puhl S., Damme A., Braunschweig H., Meinel L., Engstler M., Holzgrabe U. Synthesis and structure-activity relationships of new quinolone-type molecules against Trypanosoma brucei. J. Med. Chem. 2012;55:2538–2548. PubMed

Horvath A., Horakova E., Dunajcikova P., Verner Z., Pravdova E., Slapetova I., Cuninkova L., Lukes J. Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei. Mol. Microbiol. 2005;58:116–130. PubMed

Huang G., Vercesi A.E., Docampo R. Essential regulation of cell bioenergetics in Trypanosoma brucei by the mitochondrial calcium uniporter. Nat. Commun. 2013;4:2865. PubMed PMC

Ibrahim H.M., Al-Salabi M.I., El Sabbagh N., Quashie N.B., Alkhaldi A.A., Escale R., Smith T.K., Vial H.J., de Koning H.P. Symmetrical choline-derived dications display strong anti-kinetoplastid activity. J. Antimicrob. Chemother. 2011;66:111–125. PubMed PMC

Jannin J., Cattand P. Treatment and control of human African trypanosomiasis. Curr. Opin. Infect. Dis. 2004;17:565–571. PubMed

Jara J.A., Castro-Castillo V., Saavedra-Olavarria J., Peredo L., Pavanni M., Jana F., Letelier M.E., Parra E., Becker M.I., Morello A., Kemmerling U., Maya J.D., Ferreira J. Antiproliferative and uncoupling effects of delocalized, lipophilic, cationic gallic acid derivatives on cancer cell lines. Validation in vivo in singenic mice. J. Med. Chem. 2014;57:2440–2454. PubMed

Jensen R.E., Englund P.T. Network news: the replication of kinetoplast DNA. Annu. Rev. Microbiol. 2012;66:473–491. PubMed

Kelso G.F., Porteous C.M., Coulter C.V., Hughes G., Porteous W.K., Ledgerwood E.C., Smith R.A., Murphy M.P. Selective targeting of a redox-active ubiquinone to mitochondria within cells: antioxidant and antiapoptotic properties. J. Biol. Chem. 2001;276:4588–4596. PubMed

Kinnamon K.E., Steck E.A., Hanson W.L., Chapman W.L., Jr. In search of anti-Trypanosoma cruzi drugs: new leads from a mouse model. J. Med. Chem. 1977;20:741–744. PubMed

Kinnamon K.E., Steck E.A., Rane D.S. A new chemical series active against African trypanosomes: benzyltriphenylphosphonium salts. J. Med. Chem. 1979;22:452–455. PubMed

Koreny L., Sobotka R., Kovarova J., Gnipova A., Flegontov P., Horvath A., Obornik M., Ayala F.J., Lukes J. Aerobic kinetoplastid flagellate Phytomonas does not require heme for viability. Proc. Natl. Acad. Sci. U. S. A. 2012;109:3808–3813. PubMed PMC

Kovarova J., Horakova E., Changmai P., Vancova M., Lukes J. Mitochondrial and nucleolar localization of cysteine desulfurase Nfs and the scaffold protein Isu in Trypanosoma brucei. Eukaryot. Cell. 2014;13:353–362. PubMed PMC

Kovarova N., Mracek T., Nuskova H., Holzerova E., Vrbacky M., Pecina P., Hejzlarova K., Kluckova K., Rohlena J., Neuzil J., Houstek J. High molecular weight forms of mammalian respiratory chain complex II. PLoS One. 2013;8:e71869. PubMed PMC

La Greca F., Magez S. Vaccination against trypanosomiasis: can it be done or is the trypanosome truly the ultimate immune destroyer and escape artist? Hum. Vaccines. 2011;7:1225–1233. PubMed PMC

Lanteri C.A., Tidwell R.R., Meshnick S.R. The mitochondrion is a site of trypanocidal action of the aromatic diamidine DB75 in bloodstream forms of Trypanosoma brucei. Antimicrob. Agents Chemother. 2008;52:875–882. PubMed PMC

Liu B., Liu Y., Motyka S.A., Agbo E.E., Englund P.T. Fellowship of the rings: the replication of kinetoplast DNA. Trends Parasitol. 2005;21:363–369. PubMed

Luque-Ortega J.R., Reuther P., Rivas L., Dardonville C. New benzophenone-derived bisphosphonium salts as leishmanicidal leads targeting mitochondria through inhibition of respiratory complex II. J. Med. Chem. 2010;53:1788–1798. PubMed

Mazet M., Morand P., Biran M., Bouyssou G., Courtois P., Daulouede S., Millerioux Y., Franconi J.M., Vincendeau P., Moreau P., Bringaud F. Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability. PLoS Negl. Trop. Dis. 2013;7:e2587. PubMed PMC

Miller P.G., Klein R.A. Effects of oligomycin on glucose utilization and calcium transport in African trypanosomes. J. General Microbiol. 1980;116:391–396. PubMed

Murphy M.P. Targeting lipophilic cations to mitochondria. Biochim. Biophys. Acta. 2008;1777:1028–1031. PubMed

Nolan D.P., Voorheis H.P. The mitochondrion in bloodstream forms of Trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the F1F0-ATPase. Eur. J. Biochem. 1992;209:207–216. PubMed

Osório A.L., Madruga C.R., Desquesnes M., Soares C.O., Ribeiro L.R., Costa S.C. Trypanosoma (Duttonella) vivax: its biology, epidemiology, pathogenesis, and introduction in the New World – a review. Mem. Inst. Oswaldo Cruz. 2008;103:1–13. PubMed

Pullman M.E., Penefsky H.S., Datta A., Racker E. Partial resolution of the enzymes catalyzing oxidative phosphorylation. I. Purification and properties of soluble dinitrophenol-stimulated adenosine triphosphatase. J. Biol. Chem. 1960;235:3322–3329. PubMed

Rios Martinez C.H., Lagartera L., Kaiser M., Dardonville C. Antiprotozoal activity and DNA binding of N-substituted N-phenylbenzamide and 1,3-diphenylurea bisguanidines. Eur. J. Med. Chem. 2014;81:481–491. PubMed

Schnaufer A., Clark-Walker G.D., Steinberg A.G., Stuart K. The F1-ATP synthase complex in bloodstream stage trypanosomes has an unusual and essential function. EMBO J. 2005;24:4029–4040. PubMed PMC

Smith R.A., Hartley R.C., Murphy M.P. Mitochondria-targeted small molecule therapeutics and probes. Antioxid. Redox Signal. 2011;15:3021–3038. PubMed

Snow B.J., Rolfe F.L., Lockhart M.M., Frampton C.M., O'Sullivan J.D., Fung V., Smith R.A., Murphy M.P., Taylor K.M., Protect Study G. A double-blind, placebo-controlled study to assess the mitochondria-targeted antioxidant MitoQ as a disease-modifying therapy in Parkinson's disease. Mov. Disord. 2010;25:1670–1674. PubMed

Spivak A.Y., Keiser J., Vargas M., Gubaidullin R.R., Nedopekina D.A., Shakurova E.R., Khalitova R.R., Odinokov V.N. Synthesis and activity of new triphenylphosphonium derivatives of betulin and betulinic acid against Schistosoma mansoni in vitro and in vivo. Bioorg. Med. Chem. 2014;22:6297–6304. PubMed

Subrtova K., Panicucci B., Zikova A. ATPaseTb2, a unique membrane-bound FoF1-ATPase component, is essential in bloodstream and dyskinetoplastic trypanosomes. PLoS Pathog. 2015;11:e1004660. PubMed PMC

Swallow B.M. Food and Agriculture Organization of the United Nations; Rome: 1999. Impacts of Trypanosomiasis on African Agriculture.

Taladriz A., Healy A., Flores Perez E.J., Herrero Garcia V., Rios Martinez C., Alkhaldi A.A., Eze A.A., Kaiser M., de Koning H.P., Chana A., Dardonville C. Synthesis and structure-activity analysis of new phosphonium salts with potent activity against African trypanosomes. J. Med. Chem. 2012;55:2606–2622. PubMed

Tan T.H., Bochud-Allemann N., Horn E.K., Schneider A. Eukaryotic-type elongator tRNAMet of Trypanosoma brucei becomes formylated after import into mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2002;99:1152–1157. PubMed PMC

Teixeira J., Soares P., Benfeito S., Gaspar A., Garrido J., Murphy M.P., Borges F. Rational discovery and development of a mitochondria-targeted antioxidant based on cinnamic acid scaffold. Free Radic. Res. 2012;46:600–611. PubMed

Tielens A.G., van Hellemond J.J. Surprising variety in energy metabolism within Trypanosomatidae. Trends Parasitol. 2009;25:482–490. PubMed

Tyc J., Klingbeil M.M., Lukes J. Mitochondrial heat shock protein machinery hsp70/hsp40 is indispensable for proper mitochondrial DNA maintenance and replication. mBio. 2015;6 PubMed PMC

Vercesi A.E., Docampo R., Moreno S.N. Energization-dependent Ca2+ accumulation in Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria. Mol. Biochem. Parasitol. 1992;56:251–257. PubMed

Welburn S.C., Picozzi K., Fevre E.M., Coleman P.G., Odiit M., Carrington M., Maudlin I. Identification of human-infective trypanosomes in animal reservoir of sleeping sickness in Uganda by means of serum-resistance-associated (SRA) gene. Lancet. 2001;358:2017–2019. PubMed

Wickstead B., Ersfeld K., Gull K. Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei. Mol. Biochem. Parasitol. 2002;125:211–216. PubMed

Williams S., Saha L., Singha U.K., Chaudhuri M. Trypanosoma brucei: differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages. Exp. Parasitol. 2008;118:420–433. PubMed PMC

Wirtz E., Leal S., Ochatt C., Cross G.A. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 1999;99:89–101. PubMed

Zikova A., Schnaufer A., Dalley R.A., Panigrahi A.K., Stuart K.D. The F(0)F(1)-ATP synthase complex contains novel subunits and is essential for procyclic Trypanosoma brucei. PLoS Pathog. 2009;5:e1000436. PubMed PMC

Chelation of Mitochondrial Iron as an Antiparasitic Strategy

Mitochondrion of the Trypanosoma brucei long slender bloodstream form is capable of ATP production by substrate-level phosphorylation

Repurposing of MitoTam: Novel Anti-Cancer Drug Candidate Exhibits Potent Activity against Major Protozoan and Fungal Pathogens

Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter Trypanosoma brucei

Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite

Trypanosome Mitochondrial Translation and Tetracycline: No Sweat about Tet