Abstract
The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.
Highlights
A specific aquaglyceroporin, TbAQP2, is required for high-affinity uptake of pentamidine into human African trypanosomiasis parasites, Trypanosoma brucei [1,2,3]
To approach the question whether TbAQP2 is directly permeable for pentamidine, we set up a yeast expression system and initially analyzed permeability for small neutral and positively charged compounds
Expression of TbAQP2 did not increase the susceptibility in comparison to non-expressing and TbAQP3 expressing cells (Fig 1E)
Summary
A specific aquaglyceroporin, TbAQP2, is required for high-affinity uptake of pentamidine into human African trypanosomiasis parasites, Trypanosoma brucei [1,2,3]. At a molecular weight of 340 Da and with two strongly basic, positively charged amidine moieties (pKa 12.1), pentamidine is not a physiological substrate analog and differs from previous examples of drug uptake involving aquaglyceroporins [5,6]. In the treatment of acute promyelocytic leukemia, the human aquaglyceroporin AQP9 [7] serves as an entry site for the drug arsenic trioxide, As2O3 [8], which dissolves into weak arsenous acid, As(OH) (126 Da), resembling the glycerol molecule (92 Da). The analogous antimonous acid, Sb(OH) (173 Da), derived from the antimonial drug pentostam, is the first-line treatment of leishmaniasis and enters Leishmania major parasites via LmAQP1 [9]. The antineoplastic agent hydroxyurea (N-hydroxylated urea, 76 Da) is a permeant of PfAQP, i.e. the single aquaglyceroporin of the malaria parasite Plasmodium falciparum, and of TgAQP from the toxoplasmosis parasite Toxoplasma gondii [10]
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