Differences in Transporters Rather than Drug Targets Are the Principal Determinants of the Different Innate Sensitivities of Trypanosoma congolense and Trypanozoon Subgenus Trypanosomes to Diamidines and Melaminophenyl Arsenicals

Abstract

The animal trypanosomiases are infections in a wide range of (domesticated) animals with any species of African trypanosome, such as Trypanosoma brucei, T. evansi, T. congolense, T. equiperdum and T. vivax. Symptoms differ between host and infective species and stage of infection and are treated with a small set of decades-old trypanocides. A complication is that not all trypanosome species are equally sensitive to all drugs and the reasons are at best partially understood. Here, we investigate whether drug transporters, mostly identified in T. b. brucei, determine the different drug sensitivities. We report that homologues of the aminopurine transporter TbAT1 and the aquaporin TbAQP2 are absent in T. congolense, while their introduction greatly sensitises this species to diamidine (pentamidine, diminazene) and melaminophenyl (melarsomine) drugs. Accumulation of these drugs in the transgenic lines was much more rapid. T. congolense is also inherently less sensitive to suramin than T. brucei, despite accumulating it faster. Expression of a proposed suramin transporter, located in T. brucei lysosomes, in T. congolense, did not alter its suramin sensitivity. We conclude that for several of the most important classes of trypanocides the presence of specific transporters, rather than drug targets, is the determining factor of drug efficacy.

Nucleoside Transport and Nucleobase Uptake Null Mutants in Leishmania mexicana for the Routine Expression and Characterization of Purine and Pyrimidine Transporters

“…Kinetoplastids are useful systems for the heterologous expression of transporters. For instance, we have previously expressed the T. congolense transporter TcoAT1 in the T. brucei cell line lacking the TbAT1/P2 transporter [ 25 ], showing it did not have the proposed role in diminazene resistance [ 49 ], a conclusion recently confirmed by meticulous work on diamidine uptake in T. congolense [ 50 ]. We have also recently published the first characterization of Trichomonas vaginalis nucleoside transporters by expression in T. brucei [ 26 ] and reported a unique substrate binding mode for Toxoplasma gondii oxopurine transporter Tg244440 [ 28 ] expressed in a T. brucei cell line from which the main nucleobase transporter locus has been knocked out [ 27 ].…”

Ratiometric imaging of minor groove binders in mammalian cells using Raman microscopy

“…Please do not adjust margins Please do not adjust margins were seeded (circa 1×10 6 ) in a total volume of 2 mL onto a coverslip (22×22 mm; #1.5H) in a 6-well plate. After the overnight incubation, the media was removed and replaced with RPMI containing DMSO (control) or Alk-MGB (0.1-100 µM).…”

“…The fixation step was performed by a 4% paraformaldehyde (PFA) solution in PBS (10 mins, 37°C, 5% CO2). For the stimulated Raman scattering studies, HeLa cells were seeded (circa 1×10 6 ) in a total volume of 2 mL on a coverslip (22×22 mm; #1.5H) in a 6-well plate. After the overnight incubation, the media was removed and replaced with DMEM containing DMSO (control) or S-MGB (10 µM).…”

Purine Transporters as Efficient Carriers for Anti-kinetoplastid Molecules: 3′-Deoxytubercidin versus Trypanosomes

“…8 Curiously, despite its common use in the field to treat T. congolense infections, the uptake of diminazene in this parasite does not seem to follow a single concentrative route as in T. brucei, but it is rather mediated by several low-affinity carriers, including (a) folate and (an unknown) pentamidine transporters; 9 of note, the route for pentamidine internalization in T. congolense is also unknown, as this parasite does not possess orthologues of TbAT1 or TbAQP2. In fact, the heterologous expression of TbAT1 and TbAQP2 in T. congolense increased its susceptibility to diminazene, pentamidine, and melaminophenyl arsenicals, 10 indicating (1) that the limitation in drug internalization in T. congolense is one of the major determinants for its increased resistance to trypanocidal drugs and suggesting (2) that if a trypanocidal compound can display the same level of cellular penetration in T. brucei and T. congolense, it is likely to achieve similar trypanocidal activity in both species, except if it interferes with energy metabolism, where major metabolic differences between T. brucei and T. congolense reside. 11 This, thus, stresses the need for the finding of trypanocidal P1 permeants.…”

“…In fact, the heterologous expression of Tb AT1 and Tb AQP2 in T. congolense increased its susceptibility to diminazene, pentamidine, and melaminophenyl arsenicals, indicating (1) that the limitation in drug internalization in T. congolense is one of the major determinants for its increased resistance to trypanocidal drugs and suggesting (2) that if a trypanocidal compound can display the same level of cellular penetration in T.…”