ObjectivesTrypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs.MethodsThe TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants.ResultsAll MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical.ConclusionsTbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.

African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(Ϫ/Ϫ), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidineadapted line B48. The K m for diminazene transport in bloodstream tbat1(Ϫ/Ϫ) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(Ϫ/Ϫ), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosomes.

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

31Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to 32 pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively 33 selected for increased pore size from a common ancestor aquaporin. We demonstrate that 34 TbAQP2's unique architecture permits pentamidine permeation through its central pore and 35 show how specific mutations in highly conserved motifs affect drug permeation. Introduction 36 of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. 37 Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically 38 favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally 39 strictly impermeable for ionic species. We also identify the structural determinants that make 40 pentamidine a permeant but exclude most other diamidine drugs. Our results have wide-41 ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. 42Moreover, these new insights in aquaporin permeation may allow the pharmacological 43 exploitation of other members of this ubiquitous gene family. 44 45 Keywords: 46 Drug transport / Aquaporin / evolution of membrane proteins / Trypanosoma brucei / 47 109 endocytosis rate and the rate of pentamidine uptake. Our results unequivocally show that 110 pentamidine permeates directly through the central pore of TbAQP2 and that uptake is 111 dependent on the microbial membrane potential. Having identified the essential 112 characteristics that allow the transport of large, flexible molecules through TbAQP2, this 113 should now allow the evaluation of aquaporins in other species for similar adaptations.114 7 Results 115 116 12. Investigation of the structural determinants of AQP2 for pentamidine transport 117 1.1. Positive selection for pore size 118 In T. brucei, the AQP2 and AQP3 genes are arranged as a tandem pair on chromosome 10 119 and have 74% amino acid identity. Whereas TbAQP2 clearly mediates pentamidine uptake, 120 TbAQP3 does not (Baker et al, 2012; Munday et al, 2014), nor do various chimeric AQP2/3 121 rearrangements that give rise to pentamidine resistance (Munday et al, 2014; Graf et al, 122 2015). To investigate the origin of the AQP2 gene, a phylogenetic analysis of AQPs in 123 African trypanosomes was performed. The number of aquaporin genes varies: there is a 124 single aquaporin in T. vivax and T. congolense, two in T. suis and three in T. brucei and its 125 derivatives (Supplemental Fig. 1A). The most probable tree (Supplemental Fig. 1B) is 126 consistent with the evolutionary history of the four species (Hutchinson & Gibson, 2015) and 127 indicates AQP1 as the ancestral AQP present in all trypanosome species. A duplication 128 occurred in the common ancestor of T. suis and T. brucei after divergence from T. 129 congolense and a further duplication, to form AQP2 and AQP3, in the ancestor of T. 130 brucei after divergence from T. suis. Multiple alignment (Supplemental Fig. 1A) shows that 131 the classical NPA/NPA a...

Given the pressing need for new antiprotozoal drugs without cross-resistance with current (failing) chemotherapy, we have explored 3-tridecylpyridinium alkaloids (3TPAs), derivatives of viscosamine, as antiparasitic agents. We have developed a simple synthetic route toward viscosamine and related cyclic and linear monomers and oligomers. Evaluation for cytotoxicity on the protozoan parasites Trypanosoma brucei, Leishmania spp., and Plasmodium falciparum revealed several 3TPAs with antiprotozoal activity in the nanomolar range. Their promising selectivity index in vitro prompted us to study the dynamics of cytotoxicity on trypanosomes in more detail. Parasites were killed relatively slowly at therapeutically safe concentrations, in a process that did not target the cell cycle. Clearance of T. brucei cultures was observed at drug concentrations of 1−10 μM.