african trypanosomes are responsible for a spectrum of important human and veterinary diseases south of the sahara , transmitted by various tsetse fly species . trypanosoma brucei gambiense causes a chronic but fatal human african trypanosomiasis ( hat , or sleeping sickness ) in western and central africa , whereas t.  b. rhodesiense causes a much more acute illness in eastern and southern africa ( brun et  al . , 2010 ) ; both forms of the disease are considered almost invariably fatal if left untreated . whereas transmission of t.  b. gambiense appears to be almost exclusively anthroponotic , t.  b. rhodesiense is a zoonotic parasite , with many wild and domestic animals , particularly cattle , acting as reservoirs ( welburn et  al . , 2001 ) . in addition , trypanosoma congolense , t.  b. brucei , trypanosoma evansi and trypanosoma vivax cause animal african trypanosomiasis ( aat ) , inflicting a terrible burden on agriculture in the tsetse belt and , for the latter two species , also in areas far beyond the testse habitat including the indian subcontinent and south america ( desquesnes et  al . , 2013 , osrio et  al . , 2008 , swallow , 1999 ) . while vector control in conjunction with extensive surveillance can have highly significant local impact on the disease burden , there is no realistic prospect of a vaccine ( la greca and magez , 2011 ) and chemotherapy is practically the only option in most areas ( delespaux and de koning , 2007 , jannin and cattand , 2004 ) . however , few drugs currently exist for either hat or aat , and these are old , suffer from severe side - effects and/or resistance , and are usually only effective against certain ( sub ) species or stages of the disease ( brun et  al . , 2010 , delespaux and de koning , 2007 ) . new therapeutic agents are therefore urgently necessary , preferably active against all african trypanosome species , and against both stages of the human disease . one promising lead is the class of benzyltriphenylphosphonium compounds that displays highly potent activity against rodent models of t.  b. rhodesiense ( kinnamon et  al . , 1979 ) , trypanosoma cruzi ( kinnamon et  al . , 1977 ) and leishmania donovani ( hanson et  al . , moreover , a large series of bisphosphonium salts were shown to possess strong anti - leishmanial ( luque - ortega et  al . , 2010 ) and trypanocidal ( dardonville et  al . , 2015 , many of these compounds showed high selectivity against the kinetoplastid parasites relative to human cell lines and , importantly , displayed no cross - resistance with existing trypanocides such as diamidines and melaminophenyl arsenicals ( taladriz et  al . , 2012 ) . the triphenylphosphonium ( tpp ) moiety has been used extensively as a vehicle to deliver drugs to mitochondrial targets ( cairns et  al . , 2015 , cortes et  al . , 2015 , smith et  al . , 2011 ) . among many applications , tpp has been used to deliver functional probes ( cairns et  al . , 2015 ) , antioxidants ( kelso et  al . , 2001 , teixeira et  al . , 2012 ) , anti - cancer drugs ( jara et  al . , 2014 ) , and even liposomes ( benein et  al . , 2015 ) to mitochondria . tpp - linked natural compounds botulin and betulinic acid showed in  vitro and in  vivo activity against schistosoma mansoni ( spivak et  al . , 2014 ) . crucially , tpp - mediated mitochondrial targeting appears to be generally safe , as a double - blind , placebo controlled study with human volunteers found no evidence of side - effects of the tpp - coupled antioxidant mitoq over a 12-month period ( snow et  al . , 2010 ) . the mitochondrial accumulation of tpp - coupled drugs is driven by the strong inside - negative potential across the inner membrane of the mitochondrion . tpp , being a lipophilic cation with a highly dispersed charge is believed to diffuse freely across the inner membrane and be highly concentrated in the mitochondrial matrix , to an extent determined by the mitochondrial membrane potential m and the plasma membrane potential vm , as described by the nernst equation ( cairns et  al . , 2015 ) . showed that the anti - leishmanial tpp analogues targeted the parasite 's mitochondrion , and proposed their principal action to be the inhibition of the succinate dehydrogenase complex ( complex ii ) ( luque - ortega et  al . , 2010 ) . however , similar compounds also had strong activity against bloodstream form ( bf ) t.  brucei ( dardonville et  al . , 2015 , taladriz et  al . , 2012 ) , which has a much less elaborate mitochondrial metabolism , lacking for instance much of the krebs cycle , and the cytochrome - dependent respiratory chain ( tielens and van hellemond , 2009 ) . transfer of electrons to oxygen is instead mediated by a plant - like alternative oxidase ( chaudhuri et  al . , 1998 ) and the mitochondrial membrane potential is maintained by the fof1 atpase pumping protons from the mitochondrial matrix ( nolan and voorheis , 1992 , schnaufer et  al . , 2005 ) . we thus investigated whether the succinate dehydrogenase complex is expressed in bf t.  brucei , and whether this might be the primary target for the trypanocidal activity of triphenylphosphonium salts and their analogues . we selected two compounds with strong trypanocidal activity ( taladriz et  al . , 2012 ) to represent the main two classes of aliphatic and aromatic bisphosphonium salts ( fig .  1 ) . the effects of ahi-9 and cd38 on growth of bf trypanosoma brucei brucei strain lister 427 were investigated by incubating cultures under standard conditions ( hmi-9/10% fbs ; 37  c , 5% co2 ) in the presence or absence of 0.1  m , 0.3  m or 1  m test compound for up to 72  h. samples were taken in triplicate at the following times ( h ) after initiation of the experiment : 0 , 4 , 8 , 12 , 24 , 28 , 32 , 36 , 48 , 52 , 56 , 60 , 72 . compounds cd38 and ahi-9 were synthesized as reported previously ( luque - ortega et  al . , 2010 , fifty percent effective concentrations ( ec50 ) were determined using the fluorescence viability indicator dye alamar blue ( resazurin sodium salt , sigma ) , exactly as described ( gould et  al . , 2013 , gould et  al . , 2008 ) . serial doubling dilutions of test compounds were prepared over two rows of a 96-well plate , leaving the last well without added drug as a control , before the addition of an equal volume of cell suspension to each well . the plates were incubated for 48  h ( 37  c , 5% co2 ) before the addition of 20  l resazurin solution ( 125  g / ml in pbs ph7.4 ) per well and incubation for a further 24  h. fluorescence was determined using a fluostar optima ( bmg labtech , durham , nc , usa ) with excitation wavelength set at 544  nm and emission at 620  nm . data were plotted to a sigmoid curve with variable slope using prism 5.0 ( graphpad , san diego , ca ) . nuclei and kinetoplasts were visualized using the fluorescent dye 4,6-diamidino-2-phenylindole ( dapi ) on bf trypanosomes after fixation . 50  l of cells at 5    10  cells / ml were spread onto a glass microscope slide , left to air dry and fixed in methanol overnight at  20  c . the slides were rehydrated with 1  ml of pbs for 10  min , which was allowed to evaporate ( but not to completely dry ) . 50  l of pbs containing 1  g / ml dapi and 1% of 1,4-diazabicyclo [ 2.2.2 ] octane ( dapco ) was added to the slides and spread by coverslip . slides were observed under uv light on a zeiss axioplan microscope using a hamamatso digital camera and openlab software . 500  cells were recorded for each sample , and scored for dna configuration into the following groups : 1n1k , 1n2k , 2n2k ( early ) and 2n2k the effect of test compounds on dna configuration was determined at 0 , 8 , 12 , 16 and 24  h ; untreated cultures served as control . dna content of bf trypanosomes was measured by flow cytometry using the fluorescent dye propidium iodide ( pi ) , as described ( hammarton et  al . , 2003 , ibrahim et  al . , 2011 ) . briefly , 1  ml of cell suspension at 10  cells / ml was transferred at each time point into microfuge tubes and centrifuged at 2500  rpm for 10  min  at 4  c , then resuspended and fixed in 1  ml of 70% methanol and 30% pbs and left at 4  c overnight . the samples were washed twice with 1  ml of pbs and subsequently resuspended in 1  ml pbs containing propidium iodide and rnase a ( both at 10  g / ml ) , and incubated at 37  c for 45  min while protected from light . intracellular calcium levels were measured using the screen quest fluo-8 calcium kit ( abd bioquest , sunnyvale , ca ) . t.  b. brucei of strain lister 427 were grown in hmi-9/fcs in vented flasks incubated ( 37  c/5% co2 for 48  h ) and harvested by centrifugation ( 2500    g , 10  min  at 4  c ) , resuspended to a final density of 4    10  cells / ml and divided into aliquots for determination at 0 , 4 , 8 , 12  h of exposure to test compounds ( in hmi-9/fcs , 37  c/5% co2 ) . at an appropriate interval before the start of the measurement , aliquots of this culture were transferred to centrifuge tubes , centrifuged ( 10  min , 2500  g ) and resuspended in the fluo-8 dye - loading solution at exactly 4    10  cells / ml and further incubated at 37  c for 30  min . the cells were then washed twice with assay buffer to remove any extracellular dye - loading solution . 90  l of cell suspension was added to each well of a black - bottomed 96-well plate , and subsequently 10  l of test compound at 10    concentration was added ( except for the 0  min recording ) ; 10  m calcium ionophore a23187 ( sigma ) was used as a positive control and 10  l buffer was added to a well that served as drug - free control . the plate was then incubated in a fluostar optima fluorimeter at 37  c , and the fluorescence was observed for 10  min using cycles of 4  s. the mitochondrial membrane potential of treated and untreated cells was assessed by using tetramethylrhodamine ethyl ester ( tmre ) ( denninger et  al . the cell density was adjusted to 10  cells / ml with and without test compounds for the start of the experiment . 1  ml of sample was transferred at each time point into a microfuge tube and centrifuged at 2500  rpm for 10  min  at 4  c , and was then resuspended in 1  ml pbs containing 25  nm of tmre , and cells were incubated at 37  c for 30  min . valinomycin ( 100  nm ) and troglitazone ( 10  m ) were used as controls , as they are known to induce mitochondrial membrane depolarization and hyperpolarization , respectively ( denninger et  al . , 2007 ) . mitochondrial membrane potential was expressed as the percentage of cells displaying fluorescence higher than 100 artificial units , which was calibrated at exactly 50% for the no - drug control sample of the zero incubation time point , as described ( ibrahim et  al . , cells in the exponential growth phase were stained with 60  nm of tmre for 30  min  at 37  c . cells were pelleted ( 1300    g , 10  min , rt ) , resuspended in 2  ml of pbs ( ph 7.4 ) and immediately analysed by flow cytometry ( bd facs canto ii instrument ) . for each sample , treatment with the protonophore fccp ( 20  m ) was used as a control for mitochondrial membrane depolarization . data were evaluated using bd facsdiva ( bd company ) software . to create the sdh1 ( tb927.8.6580 ) rnai construct , a 485 bp fragment was pcr amplified from t.  brucei strain 427 genomic dna with the following oligonucleotides : fw : gat gga tcc ctc tgg gct tcg tgc cgc aa , rev : gga aag ctt tgc cac gac aac agc cgt cc utilizing the respective bamhi and hindiii restriction sites inherent in the primers ( underlined ) . the digested amplicon was then cloned into the p2t7 - 177 plasmid ( wickstead et  al . , 2002 ) . this plasmid was used for transfection of pf t.  brucei lister 427 29.13 and bf lister 427 single marker ( sm ) t.  brucei cells that are transgenic for the t7 rna polymerase and the tetracyclin ( tet ) repressor ( wirtz et  al . pf 29.13  cells were grown in  vitro at 27  c in sdm79 medium containing hemin ( 7.5  mg / ml ) , hygromycin ( 25  g / ml ) , g-418 ( 10  g / ml ) and 10% foetal bovine serum . bf sm cells were grown in  vitro at 37  c in hmi-9 medium containing g-418 ( 2.5  g / ml ) and 10% foetal bovine serum . after the transfection , both cell lines were selected using phleomycin ( 2.5  g / ml ) . the inducible expression of double - stranded rna was triggered by the addition of 1  g / ml tetracycline to the medium . growth curves were generated by measuring the cell density of tet - treated and untreated cultures using the z2 cell counter ( beckman coulter inc . ) . throughout the experiment , pf and bf cultures were split daily to ensure they continuously maintained an exponential growth phase of 10 - 10  cells / ml and 1010  cells / ml , respectively . , 2012 , subrtova et  al . , 2015 ) and the mitochondria were lysed using digitonin at detergent : protein ratio of 4  mg:1  mg . high resolution clear native ( hrcn ) page analysis was performed by fractionating 50  g of mitochondrial lysate on a 312% clear native ( cn ) page gel followed by a protein transfer onto a nitrocellulose membrane . protein samples from mitochondrial lysates were also separated on sds page gels and blotted onto a pvdf membrane . both membranes were incubated with a specific sdh1 peptide antibody ( 1:1000 ) ( koreny et  al . , 2012 ) , followed by an incubation with a secondary hrp - conjugated anti - rabbit antibody ( 1:2000 , biorad ) . the specific reaction was visualized using the clarity western ecl substrate ( biorad ) on a chemidoc instrument ( biorad ) . the specific sqr activity was measured in a crude mitochondrial preparation ( 15g ) in 1  ml of sdh buffer ( 25  mm  k2hpo4 , ph 7.2 , 5  mm mgcl2 , 20  mm sodium succinate ) containing specific inhibitors of respiratory complexes iii and iv ( 0.0002% antimycin , 2  mm kcn ) and 50m dcip ( 2,6-dichlorophenolindophenol ( sigma ) ) , which acts as the electron acceptor . the unit of succinate  dehydrogenase activity ( u ) is defined as an amount of enzyme in 1  mg of kinetoplastid proteins , which causes the conversion of 1  nmol of dcip in 1  min , as monitored at 600  nm ( birch - machin and turnbull , 2001 ) . mitochondrial vesicles were purified as described previously ( acestor et  al . , 2011 ) . afterwards they were resuspended in buffer a ( 0.25  m sucrose , 50  mm tris hcl ph 8 , 1  mm dtt ) and the suspension was sonicated on power 8.5 for 3    40  s , with an incubation on ice of 1  min between sonications to prevent heating the sample . the samples were further sonicated for 10 15-s  bursts , again left on ice for 1  min between the sonications ( sonicator 3000 , misonix , 19  mm probe , 250  w ) . the submitochondrial particles were then pelleted overnight in a sw60ti rotor at 52,000  g  at 4  c . the smp pellet was resuspended in buffer b ( 0.25  m sucrose , 50  mm tris hcl ph 8.0 , 1  mm dtt , 4  mm edta , 2  mm adp ) . chloroform , saturated with 2  m tris  hcl ph 8.5 , was added to the pellet and then vigorously shaken for 20  s followed by immediate centrifugation for 5  min at 8500  rpm  at room temperature . the top ( light brown ) layer was taken , leaving the middle ( dark brown ) and the bottom ( chloroform containing ) layers behind . the f1 depleted smps and residual chloroform were removed by centrifugation in a sw28 rotor at 27,000  rpm for 30  min at room temperature , using chloroform resistant polyallomer tubes . the f1-atpase containing supernatant was carefully removed to avoid any chloroform contamination , blown with nitrogen for 15  min to remove all traces of chloroform . the f1-atpase was filtered , concentrated down to 750  l and loaded on a superdex 200 10/300  gl . the f1-atpase was purified on a fplc using a specific f1-column buffer ( 20  mm tris ph 8.5 , 200  mm nacl , 1  mm dtt , 4  mm edta , 1  mm adp , 0.002% pmsf ) . the amount of atp hydrolysis in a crude mitochondrial preparation or by affinity purified f1 atpase was measured by atp regenerating assay ( pullman et  al . , 1960 ) that couples the hydrolysis of atp with the oxidation of nadh . crude mitochondrial lysates ( 15  g ) or purified f1 atpase ( 0.7  g ) were incubated with different concentrations ( 5200  m ) of cd38 and ahi-9 for 5  min after which the mix was transferred to a 1-ml uv cuvette containing an atpase buffer ( 50  mm tris , ph 8 , 50  mm kcl , 2  mm mgso4 , 200  m nadh , 1  mm phosphoenol pyruvate , 5  l of lactate dehydrogenase ( sigma ) and 9  l of pyruvate kinase ( sigma ) ) . the reaction was then initiated by adding 2  mm mgatp ( from 100 solution of 400  mm tris , 200  mm mgso4 , 200  mm atp ) and the change of absorbance at 340  nm was measured for 5  min . azide ( 1  mm ) was used as a known inhibitor of f1 atpase activity ( bowler et  al . , 2006 ) . specific atpase activity of mitochondrial lysate or of purified f1 atpase was calculated from the amount of nadh oxidized in 1  min , and normalised to the amount of the protein in the sample . atp production in digitonin - extracted mitochondria was measured following a protocol described previously ( allemann and schneider , 2000 ) . briefly , crude mitochondrial fractions from the rnai knockdown cell lines were obtained by digitonin extraction ( tan et  al . , 2002 ) . atp production in these samples was induced by the addition of 67  m adp and 5  mm of succinate . the mitochondrial preparations were preincubated for 10  min on ice with the inhibitors malonate ( 6.7  mm ) and atractyloside ( 33  g / ml ) . the concentration of atp was determined by a luminometer ( orion ii , berthold detection systems ) using the cls i atp bioluminescence assay kit ( roche applied science ) . spr experiments were performed at 25  c with a biacore x100 apparatus ( ge healthcare , biacore ab , uppsala , sweden ) in mes buffer ( 10  mm 2-(n - morpholino)ethanesulfonic acid , 1  mm edta , 100  mm nacl , 0.005% surfactant p20 , ph 6.25 ) . the 5-biotin labelled dna hairpins 5-cgaattcgtctccgaattcg-3 and 5-cgcgcgcgttttcgcgcgcg-3 ( the loop is underlined ) were purchased from sigma the spr measurements were carried out as described ( rios martinez et  al . , 2014 ) . we investigated the actions of two bisphosphonium compounds on bloodstream form ( bf ) t.  b. brucei . the compounds cd38 and ahi-9 were selected as ( i ) displaying sub - micromolar activity against the parasite , ( ii ) possessing > 300-fold in  vitro selectivity with a human cell line and ( iii ) representing the two main classes of phosphonium trypanocides , possessing either linear saturated aliphatic substituents ( n - pentyl ; cd38 ) or aromatic substituents ( phenyl and 3-methylphenyl ; ahi-9 ) on the phosphonium moiety ( taladriz et  al . , 2012 ) . both compounds were confirmed to be active against t.  brucei , with sub - micromolar activity against bf cells and about an order of magnitude less activity against the procyclic forms ( pf ) ( table  1 ) . at concentrations up to the ec50 values the compounds caused delayed growth rather than cell death and even at concentrations several fold above ec50 ( 1  m ) the initial effect appeared to be immediate ( cd38 ) or slightly delayed ( ahi9 ) growth arrest , followed by cell death 1624  h later ( fig .  2 ) . we thus investigated whether the compounds induced cell cycle arrest in a specific phase of the cycle , using flow cytometry to measure the dna content in single cells of the population . as expected , we found that untreated control cells were less prone to enter s - phase when the culture entered the late log - phase of growth , leading to a small increase in g1-phase cells after 16  h ; the proportion of g1-phase cells had initially decreased as the culture progressed from stationary to early log phase ; the proportion of cells in s - phase steadily declined during late log phase ( fig . similar effects , but much more pronounced and at much lower cell densities , were observed in the cultures treated with cd38 or ahi-9 : both displayed a steep decline in s - phase cells after 8  h , accompanied by a significant increase in g1-phase cells ( fig . 3a , b ) , although the cultures were in virtual growth arrest and at comparatively low density ( fig . we thus conclude that bisphosphonium compounds prevent trypanosomes from entering the cell cycle and initiating dna synthesis . cd38 also caused a highly significant decrease in cells in the first stages of trypanosomal cell division , i.e. with 1 nucleus and 2 kinetoplasts ( 1n2k ) , or with 2 of each ( 2n2k ) but no cleavage furrow initiated ( 2n2k - early ) , after 8  h of treatment ; the percentage of cells in all stages of division ( in2k , 2n2k ) continued to decline sharply with the exposure time ( fig . this experiment showed that bisphosphonium - treated cells were unable to replicate kinetoplast dna and/or initiate kinetoplast division . it was not possible to reliably distinguish between the two possibilities , although we did not observe increased dapi fluorescence associated with the kinetoplast ( i.e. kdna content ) after cd38 treatment ( not shown ) ; however , it is uncertain whether this would have been sufficiently unambiguous . however , it has previously been reported that cd38 does not bind directly to a poly(da.dt)2 dna polymer ( dardonville et  al . , 2006 ) , and we here follow that up with spr measurements of cd38 and ahi-9 binding to two other dna . neither compound showed measurable binding to the cgaattcg oligonucleotide , and only cd38 interacted , weakly , with the cgcgcgcg sequence , with a kd of 7.0    10  m ( supplementary fig . it has been reported that cd38 elicits anti - leishmanial activity by inhibiting the succinate dehydrogenase complex ( also known as complex ii ) in the l. donovani mitochondrion ( luque - ortega et  al . , 2010 ) , and a mitochondrial target would be consistent with the observed phenotype of being unable to initiate kinetoplast division ( above ) . incubation of bf t.  brucei with cd38 or ahi-9 for 4  h , at concentrations close to their respective ec50 values , caused a dose - dependent reduction of the mitochondrial membrane potential m ( fig . were observed after just 30  min ( p  <  0.05 for cd38 ) and much more dramatic reductions were seen after 4  h of exposure ( p  <  0.001 for both compounds ) until the mitochondrial membranes appeared to be almost fully depolarized ( fig . this was accompanied by a similar drop in cellular atp levels , which was highly significantly different from control ( p  <  0.01 ) and was statistically identical to treatment with the f1fo atpase inhibitor oligomycin after 1  h. this level of cellular atp remained essentially constant for at least 12  h of treatment with either of the test compounds ( fig . the decrease of cellular atp is in agreement with the observed redirection of glycolysis to 1 molecule of glycerol and 1 molecule of pyruvate due to m dissipation ( see the discussion ) . another cellular effect was that after four hours of incubation with cd38 or ahi-9 the level of free intracellular ca started to increase ( fig . 4d ) , which was consistent with the depolarized mitochondria being unable to maintain their capacity as calcium reservoir ( huang et  al . , 2013 ) . because the target of bisphosphonium salts in l. donovani cells was suggested to be the succinate dehydrogenase ( sdh ) complex ( luque - ortega et  al . , 2010 ) , we explored , in both pf and bf t.  brucei , its steady state protein level , the assembly of the complex , and whether its depletion elicits a reduced growth rate . first , we determined the steady state levels of a specific subunit of the sdh complex , sdh1 , which contains the cofactor fad and is responsible for the electron entry into the complex . when mitochondrial lysate was fractionated on a tgx stain - free precast gel followed by western blot analysis using anti - sdh1 antibody , the correct size band for sdh1 ( 66  kda ) was detected . the highest level of expression is seen in pf cells and much lower amounts of sdh1 subunit are detected in bf cells ( fig .  5a , upper panel ) . equal loading of protein samples was verified by uv detection of proteins ( fig .  5a , lower panel ) . independently - generated mitochondrial samples were analysed repeatedly with similar results , confirming the relatively low intensity of the sdh1 signal in bf cells compared to pf , and clearly show a much lower steady - state sdh1 protein level in the former life cycle stage . to gain better insight about the significance of the function of the sdh complex in pf and bf cells , the level of assembled sdh complex was analysed by high resolution clear native ( hrcne ) page . 2011 ) is prominent in the pf sample , but only a very faint band is detected in bf mitochondrial lysate ( fig . 5b ) , which correlates well with the relative steady state levels of sdh1 in the respective life stages of t.  brucei . interestingly , two bands were detected in the pf sample , suggesting that the complex may form higher molecular weight aggregates , or associate with another macromolecular structure , as suggested by kovarova et  al . the activity of the sdh complex can be followed spectrophotometrically as a change in the absorbance of dcip , with electrons flowing from succinate to dcip via ubiquinone q2 . each cell line was also incubated with the specific sdh inhibitor malonate to verify that only this specific activity was detected . the succinate - ubiquinone reductase ( sqr ) activity measured in bf cells corresponded to only 7% of the activity of sqr measured in pf cells ( table  2 ) . these data closely correspond with the relative steady state protein levels of sdh complex ( see above ) , and confirm that the sdh complex is much more abundant and is thus likely to play a more important role in the mitochondria of pf than bf cells . it has been shown that the sdh complex possesses an essential function in pf t.  brucei ( strain eatro1125 ) when grown in the absence of glucose , while its function was unimportant for cells grown in the presence of glucose ( coustou et  al . , nevertheless , both tested compounds exert a cytotoxic effect for pf 427 lister strain t.  brucei grown in the presence of glucose ( table  1 ) . to address whether the sdh complex is essential for the viability of this specific strain of t.  brucei , and under the specific conditions used in our study , a cell line expressing rnai targeting the sdh1 subunit was generated . when pf sdh1 rnai cells were grown in glucose - rich media , no significant difference in growth of non - induced and tetracycline - induced rna cells was observed . 5c ) confirming that the sdh complex is not essential for pf cells under glucose - rich growth conditions . to test whether the sdh complex is important for the viability of bf t.  brucei cells , 5d ) . to verify successful depletion of sdh1 protein in the pf and bf rnai induced cell lines , a western blot analysis was performed with the specific sdh1 antibody . in pf rnai tetracycline - induced cells a band of the appropriate size had disappeared after 1 day , but a band of somewhat lower apparent weight remained unchanged ( fig . in order to confirm that the upper band was specific for sdh1 , the tetracycline was washed out from the media ( fig .  5e , -tet samples ) which led to the reappearance of the upper band within 48  h. in bf sdh1 rnai cells , a single distinct band of the expected size was observed in non - induced cells ( n ) , but this band was undetectable after just 24  h of rnai induction ( fig . furthermore , the specific sqr activity of sdh complex in pf rnai sdh1 cell line was decreased by 62% after 1 day of rnai induction , and almost completely absent by day 3 ( table  3 ) . the mitochondrial membrane potential in bf cells is maintained exclusively by the hydrolytic activity of the fof1 atpase complex ( schnaufer et  al . , 2005 , subrtova et  al . , 2015 ) , while in pf cells the proton motive force is generated by the respiratory chain complexes ( gnipova et  al . in order to determine whether the depletion of sdh complex has any effect on the mitochondrial membrane potential in pf cells , the electrochemical gradient was measured using a flow cytometry analysis with the dye tmre that accumulates only in charged mitochondria . the membrane potential remained unchanged after even 5 days of rnai induction ( fig . 5 g ) indicating that the sdh complex does not significantly contribute to m in pf t.  brucei cells . the reductions in m and cellular atp levels are the early effects seen after the treatment of t.  brucei with bisphosphonium compounds ( see above ) . however , we here show that , in t.  brucei , the sdh complex is not essential and does not contribute to the membrane potential . it seems therefore unlikely that the principal trypanocidal activity of bisphosphonium salts would be on this complex and we thus explored other potential mitochondrial targets - in particular , the fof1 atpase complex that maintains m in bf cells . first , in  vitro atpase assays were performed using a purified mitochondrial fraction , which was lysed by digitonin . the prepared lysed mitochondrion - enriched fractions from pf cells were treated with various concentrations of cd38 ( fig . since there are many atpases in these samples , the fractions were also treated with the f1-atpase inhibitor azide . typically , 1  mm azide inhibits 3040% of all measured atp hydrolysis activity in such samples ( zikova et  al . , 2009 ) . increasing concentrations of either drug elicited a similar ( cd38 ) or identical ( ahi-9 ) level of inhibition as the azide control , albeit at much higher concentrations than their measured ec50 values . these inflated concentrations might be an artifact of the in  vitro assay performed on lysed instead of intact and charged mitochondria , as the latter would be able to concentrate the drugs , driven by m . importantly , the same level of inhibition was observed when the crude mitochondria were treated with azide together with a bisphosphonium salt , indicating that the inhibitions were not additive and both inhibitors were therefore acting on the same target . furthermore , the enzymatic activity was more strongly affected by ahi-9 , which typically has a stronger effect on trypanosome cells than cd38 . to further evaluate the mechanism of action of bisphosphonium salts , we determined whether these compounds also affect the rate of atp synthesis by fof1-atp synthase . this experimental set - up relies on a complete and functional oxidative phosphorylation pathway and is performed with intact , charged mitochondria isolated from pf cells ( allemann and schneider , 2000 ) . malonate and atractyloside are used as positive controls , as the former inhibits the succinate dehydrogenase complex ( i.e. the entry of the electrons ) , and the latter inhibits the atp / adp carrier ( i.e. the supplier of the substrate ) . not surprisingly , the results indicated that both cd38 and ahi-9 dose - dependently inhibited atp production in intact t.  brucei mitochondria , with concentrations similar to the measured ec50 values moderately inhibitory and higher concentrations inhibiting atp production almost completely ( fig . 7 ) . while this assay does not rule out succinate dehydrogenase as a potential target , the dose - dependency shows unambiguously that an active mitochondrial m is important to these in  vitro assays , as the test compounds were much more active than on mitochondrial lysates . since the effects on total mitochondrial atp hydrolytic and atp synthetic activities were indicative of specific inhibition of the fof1 atp synthase / atpase , we repeated the assay on highly purified f1-atpase , and observed a similar dose dependency of inhibition as above on the lysed mitochondrial fractions , i.e. less potent than in intact and charged mitochondria . in this experiment , azide inhibits almost 100% of the activity of the purified f1-atpase , confirming the purity of the sample . the increasing concentrations of the bisphosphonium test compounds reduced the atp hydrolysis activity in a linear manner . in fact , 200  m ahi-9 inactivated f1-atpase almost to the same extent as azide ( fig . we conclude that inhibition of the f1-atpase is at a minimum an important contributing factor in the mechanism of action of bisphosphonium compounds on t. brucei . the phosphonium salts were discovered as potent chemotherapeutics against trypanosoma cruzi and t.  b. rhodesiense in the late 1970s ( kinnamon et  al . , 1977 , thirty years later benzophenone - derived bisphosphonium salts were synthesized showing much higher potency against leishmania parasites . some of these phosphonium derivatives act as lipophilic cations with enhanced hydrophobicity that allow them to cross biological membranes and accumulate in organelles with a strong electrochemical gradient , e.g. mitochondria . interestingly , in mammalian cells , these lipophilic phosphonium ions are being developed to deliver antioxidants or anti - cancer drugs to mitochondria as they pass easily through the lipid bilayers . because of their disperse charged the electrochemical gradient drives their accumulation into the mitochondrial matrix concentrating them as high as 100500 fold ( murphy , 2008 ) . the nature of the trypanocidal activity of these compounds has not been determined yet , but in l. donovani some bisphosphonium compounds including cd38 were shown to have a negative impact on proper mitochondrial function ( luque - ortega et  al . , 2010 ) . upon treatment with a low drug concentration ( 0.7  m ) the mitochondria were swollen , the mitochondrial m was significantly decreased , the oxygen consumption rate , using succinate as a substrate , was affected , and the cellular atp levels were reduced . these results would suggest that the drug was either affecting a component of the oxidative phosphorylation pathway , or was acting as a kind of ionophore or charge carrier , depolarizing the mitochondrial membrane . the study proposed that in l. donovani the bisphosphonium compounds act through inhibition of the respiratory complex ii , succinate dehydrogenase ( luque - ortega et  al . while the available data certainly suggest that cd38 might be able to inhibit succinate dehydrogenase in leishmania , it seems unlikely that this would be the mode of action for bisphosphonium drugs in t.  brucei . since metabolic activities of bf mitochondria are largely repressed with the exception of lipid metabolism ( guler et  al . , 2008 ) , ion homeostasis ( nolan and voorheis , 1992 ) , calcium signalling ( huang et  al . , 2013 , 1992 ) , fes cluster assembly ( kovarova et  al . , 2014 ) and acetate production for de novo lipid biosynthesis ( mazet et  al . , 2013 ) , it is generally assumed that succinate dehydrogenase complex is largely inactive and unimportant for these cells . therefore , we decided to investigate how bisphosphonium salts interact with the unique metabolism of bf trypanosomes . we found that adding cd38 and ahi-9 , at concentrations close to their trypanocidal ec50 values , into the t.  brucei culture prevents the initiation and replication of kdna , reduces cellular atp levels , and depolarizes the mitochondrial membrane potential within a few hours of treatment ; this was immediately followed by increased cytosolic ca levels . all these phenotypic changes point also to a mitochondrial target , although not necessarily to succinate dehydrogenase . succinate dehydrogenase does not directly contribute to the membrane potential by pumping protons , as it passes its electrons to ubiquinone . moreover , we show that in bf it is not essential , as rnai knockdown presented no growth phenotype , and indeed succinate dehydrogenase is barely present as an assembled complex in this life - cycle stage . furthermore , the succinate dehydrogenase complex is also not essential for pf t.  brucei grown under high glucose conditions ( this work and ( coustou et  al . , 2008 ) ) and does not contribute to the mitochondrial membrane potential  yet , both of the tested drugs have a strong effect against pf cells ( ec50 values in single digit m range ) and decreased the mitochondrial m . we thus conclude that , in trypanosomes , inhibition of succinate dehydrogenase is not mode of action of the bisphosphonium compounds . interestingly , the potent trypanocidal diamidine db75 induces a similar phenotype in bf cells , as its treatment collapses the mitochondrial m and inhibits glucose - dependent cellular respiration , apparently through inhibition of the hydrolytic function of fof1 atpase ( lanteri et  al . , 2008 ) , which , in bf cells , employs its reverse function in order to maintain the mitochondrial m ( schnaufer et  al . , 2005 , these actions of db75 certainly overlap with those of the specific fof1 atpase inhibitor oligomycin , which decreases mitochondrial membrane potential , inhibits oxygen consumption and reduces cellular atp levels by 50% ( nolan and voorheis , 1992 ) . we observed similarly reduced cellular atp levels upon treatment with the bisphosphonium compounds cd38 and ahi-9 . the observed reduction in atp is consistent with inhibition of the fof1 complex , which is followed by immediate mitochondrial membrane depolarization , as in bf cells respiration through alternative oxidase is sensitive to m dissipation ( miller and klein , 1980 ) . the membrane depolarization in turn results in the loss of the glycerol-3-phosphate shuttle and the subsequent redirection of glycolysis to 1 molecule of glycerol and 1 molecular of pyruvate . thus , pyruvate production falls by 50% upon oligomycin treatment ( nolan and voorheis , 1992 ) and only 1 molecule of atp is made from 1 molecule of glucose . similarly , we report an approximately 50% reduction in cellular atp concentration upon treatment with bisphosphonium drugs . moreover , our data strongly suggest that bisphosphonium salts cd38 and ahi-9 inhibit the hydrolytic activity of the fof1 atpase in  vitro . inhibition assays , performed on mitochondrial lysates from pf cells and on highly purified f1-atpase , showed highly significant inhibition of atpase activity to similar levels as achieved with 1  mm azide . furthermore , a combination of azide plus cd38 or ahi-9 did not result in additional inhibition of atpase activity compared to either drug separately , suggesting that no other mitochondrial atpases play a significant role in the observed phenotype . interestingly , all the biochemical analyses performed revealed a greater sensitivity to ahi-9 than to cd38 , which correlates nicely with the almost 4-fold lower ec50 value for the former compound . it should be noted that the in  vitro assay , which measured atpase activity in lysates , required concentration of the compounds that are considerably higher than the ec50 values , as the lipophilic cations greatly concentrate in the mitochondrion ( murphy , 2008 ) , because m creates a negative charge in the matrix . this assumption is completely consistent with our results from the atp production assay , which utilizes intact and charged mitochondria from pf cells . in this assay , atp production was inhibited by the bisphosphonium compounds at the concentration similar to ec50 values . the reduction in cellular atp levels may be the immediate cause of the observed failure in initiating the s - phase of the cell cycle ( dna synthesis ) . trypanosomatid kdna is a highly organized structure composed of thousands of minicircles and tens of maxicircles ( liu et  al . , 2005 ) . to ensure that daughter cells receive completely identical kdna networks , the replication process is highly synchronized with nuclear replication followed by cell division ( jensen and englund , 2012 ) . kdna replication relies on atp and hundreds of mitochondrial proteins ( beck et  al . , 2013 ) , which are imported to mitochondria via the m - dependent tim machinery ( williams et  al . , 2008 ) . thus it is plausible to speculate that a lack of atp and decrease of m will interfere with proper initiation of kdna replication and result in cell cycle arrest . similar results were observed upon rnai knock - down of the atp / adp carrier in pf t.  brucei cells , in which reduced atp levels led to failure of the overall cell division machinery ( gnipova et  al . , 2015 ) . in contrast , a direct inhibition by bisphosphonium salts of kdna replication , segregation and maintenance as their primary mode of action is far less likely , as ( i ) these compounds have a poor affinity to dna as shown previously ( dardonville et  al . , 2006 ) and in this study , ( ii ) cd38 and ahi-9 cause a decrease in m in t.  brucei cells that lack mitochondrial dna ( gould , schnaufer and de koning , unpublished ) , and ( iii ) interference with kdna replication , either by rnai or drug treatment , usually leads to complete loss of kdna ( bruhn et  al . , 2011 , , our data strongly suggest that bisphosphonium compounds cd38 and ahi-9 primarily act on trypanosomes through inhibition of the f1-atpase although additional mitochondrial targets can not be excluded .
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 .