Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2

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.

Fig 1. Cation exclusion by TbAQP2.

(A) Shown is the layout of the aromatic/arginine selectivity filter of TbAQP2 (left) and TbAQP3 (right) as seen from the top. A bound glycerol molecule is shown as spheres. The models are based on the E. coli GlpF structure (PDB# 1FX8). (B) Western blot using membrane protein fractions isolated from yeast expressing TbAQP2 and TbAQP3 that were N-terminally tagged with a hemagglutinin epitope. (C) Phenotypic growth assay of 31019bΔmep1-3 yeast lacking endogenous ammonium transporters on media with NH₄ ⁺ as the sole nitrogen source. Growth at pH 6.5 indicates mainly background transmembrane diffusion of uncharged NH₃; growth at pH 5.5 is due to transport of charged NH₄ ⁺. LeAMT1;1 is a tomato ammonium transporter, cells without transporter expression (–) served as negative controls. With proline as a nitrogen source growth is even for all cells. (D) Complementary phenotypic growth assay using yeast-toxic methylammonium instead of ammonium yielding opposite effects. (E) Expression of TbAQP2 does not increase the pentamidine-susceptibility of yeast grown on media with glycerol (YPG) as a non-fermentable carbon source. (F) Biophysical light scattering assays with TbAQP2-expressing yeast protoplasts in 300 mM hypertonic solute gradients. The rapid first phase of the traces indicates water efflux, followed by a slower solute influx phase. Red traces indicate permeability for physiological substrates glycerol (left) and urea (right). Traces of control cells without TbAQP2 are black. Structures and traces of 3-amino-propane-1,2-diol (left) and formamidine (right) are labeled dark blue (pH 7.2) or light blue (pH 9.2). The insets show deduced permeability coefficients (P_sol) with S.E.M. from three independent experiments.

Fig 2. Inhibition of TbAQP2 glycerol permeability by pentamidine and derivatives.

(A) Shown are example traces of protoplast light scattering in 300 mM isotonic glycerol gradients in the presence of pentamidine. Red traces indicate uninhibited glycerol influx via TbAQP2 (left) and TbAQP3 (right), effects of pentamidine concentrations from 50 nM to 50 μM are colored light blue, and of 500 μM pentamidine in dark blue. (B) Structure-function evaluation of TbAQP2 inhibition by pentamidine. The chemical structures of the used compounds are shown on the left and respective dose-response curves on the right. (C) Effect of pH titration of TbAQP2 Asp265 and replacement by mutation to alanine on pentamidine inhibition. The dashed lines show the pentamidine inhibition of wild-type TbAQP2 at pH 7.2 (data taken from Fig 2B). The left panel shows dose-response curves of pentamidine pH 3.5 (closed symbols; IC₅₀ 450 nM), and pH 2.5 (open symbols; IC₅₀ 1.1 μM). The effect of the Asp265Ala point mutation (expression confirming Western blot in the inset) on the inhibition by pentamidine is depicted on the right. Data taken at pH 7.2 (IC₅₀ 4.5 μM) are indicated by closed symbols, those at pH 2.5 (IC₅₀ 5.8 μM) by open symbols. Each data point is an average of 5–9 light scattering traces each from at least two independent experiments.

Fig 3. Loss of pentamidine inhibition and gain of resistant parasites by a TbAQP Leu264Arg mutation.

(A) Light scattering assay with yeast protoplasts expressing TbAQP2 Leu264Arg in a 300 mM hypertonic glycerol gradient. Data taken in the absence of pentamidine are labeled red, those taken in the presence of 50 μM pentamidine are blue, and protoplasts without an AQP are black. The error bars denote S.E.M. from three independent experiments. (B) Western blot analysis showing inducible expression of GFP-TbAQP2 Leu264Arg (^GFPAQP2^L264R). (C) Immunofluorescence microscopy analysis of T. brucei expressing ^GFPAQP2^L264R. Scale-bar, 5 μm. (D) Dose–response curve for pentamidine in aqp2-null stains re-expressing wild-type GFP-TbAQP2 or ^GFPAQP2^L264R. EC₅₀ values are indicated. Error bars indicate SD from two independent clones assessed in triplicate assays.

Fig 4. Model of the pentamidine binding mode to TbAQP2 and proposed uptake by endocytosis in the flagellar pocket.

(A) Shown are the crystal structure of the prototypical aquaglyceroporin GlpF and a model of TbAQP2. GlpF Arg206 and TbAQP2 Leu264 mark the position of the ar/R selectivity filter. In TbAQP2, the Asp265 sidechain carboxylate binds to an amidine moiety of pentamidine (light blue), whereas in GlpF the space is occupied by the guanidine sidechain of Arg206. The location of the ‘NPA/NPA’ region (white bar) and sequence deviations in TbAQP2 are indicated. (B) Proposed uptake mechanism of pentamidine via high-affinity binding to TbAQP2, endocytosis of the complex, and release of pentamidine in the acidic lysosome due to pH shift or TbAQP2 degradation.