Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

Abstract

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.

Pharmacokinetic Comparison To Determine the Mechanisms Underlying the Differential Efficacies of Cationic Diamidines against First- and Second-Stage Human African Trypanosomiasis

“…In addition, several trypanosomal transporters have been implicated in the uptake of cationic diamidines into trypanosomes. These include the P2 aminopurine transporter (TbAT1) (35)(36)(37) and aquaglyceroporin 2 (38). The P2 aminopurine transporter belongs to the nucleoside transporter family.…”

High-Throughput Screening Platform for Natural Product–Based Drug Discovery Against 3 Neglected Tropical Diseases: Human African Trypanosomiasis, Leishmaniasis, and Chagas Disease

“…Values obtained were similar to those previously reported. 14,19,20 To determine the optimal extract concentration to obtain a reasonable hit rate, different extract dilutions were tested for each parasite. The 2 main factors considered when establishing the optimum extract concentrations were that (1) the crude extracts to be tested were prepared in 20% DMSO and that cytotoxic concentrations of this solvent should be avoided; and (2) a hit rate of about 2-5% is desirable, a hit being defined as an extract that, at a given dilution, exhibits a growth inhibition of ≥ 70%.…”

Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species

“…In fact, T. brucei gambiense field isolates with reduced susceptibility to melarsoprol harbor TbAQP2 mutations and gene loss (19). A similar functional correlation was demonstrated in L. mexicana, in which the heterologous expression of TbAQP2 increased the sensitivity of arsenicals Ͼ1,000-fold (20).…”