Experimental Evaluation of Proposed Small-Molecule Inhibitors of Water Channel Aquaporin-1

Abstract

The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl2 inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 µM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority.

Fig. 1.

Chemical structures of putative AQP1 inhibitors. (A) Structures of inhibitors reported by Migliati et al. (2009), Mola et al. (2009), Yool et al. (2013), Seeliger et al. (2013), To et al. (2015), and Patil et al. (2016) (see Table 1). (B) Osmotic water permeability in human erythrocytes as measured from the time course of scattered light intensity in response to a 250 mM inwardly directed sucrose gradient at room temperature. Erythrocytes were incubated with 0, 75, 150, or 300 µM HgCl₂ for 5 minutes before measurement.

Fig. 2.

Osmotic water permeability in human erythrocytes and hemoglobin-free ghost membranes derived therefrom. Osmotic water permeability was measured from the time course of scattered-light intensity in response to a 250 mM inwardly directed sucrose gradient. (A) Representative time course data for negative control (0.5% DMSO vehicle alone), 0.3 mM HgCl₂ (positive control), and indicated compounds (each 50 μM). Cells and ghosts were incubated with test compounds for 15 minutes before measurements. (B) Relative osmotic water permeability (S.E., n = 4). *P < 0.05 compared with control.

Fig. 3.

Osmotic swelling of human erythrocytes. Osmotic water permeability was measured from the time course of scattered-light intensity in response to a 150 mOsm outwardly directed osmotic gradient. (A) Representative time course data for negative control (0.5% DMSO vehicle alone), 0.3 mM HgCl₂ (positive control), and indicated compounds (each 50 μM). (B) Relative osmotic water permeability (S.E., n = 4). *P < 0.05 compared with negative control (0.5% DMSO vehicle alone). Cells and ghosts were incubated with test compounds for ∼15 minutes before measurements.

Fig. 4.

Osmotic swelling of calcein-labeled human erythrocytes. Osmotic water permeability was measured from the time course of intracellular calcein fluorescence in response to a 150 mOsm outwardly directed osmotic gradient. (A) Representative time course data for negative control (0.5% DMSO vehicle alone), 0.3 mM HgCl₂ (positive control), and indicated compounds (each 50 μM). (B) Relative osmotic water permeability (S.E., n = 4). *P < 0.05 compared with negative control (0.5% DMSO vehicle alone).

Fig. 5.

Compound effects on hemoglobin release and erythrocyte morphology. (A) Hemoglobin release from human erythrocytes after 15 minutes of incubation with test compounds at 50 µM (S.E., n = 4). *P < 0.05 compared with control. (B) Representative phase-contrast photomicrographs of human erythrocytes after 15-minute incubations with test compounds.

Fig. 6.

Osmotic water permeability in rat erythrocytes and hemoglobin-free ghost membranes derived therefrom. Osmotic water permeability was measured from the time course of scattered light intensity in response to a 250 mM inwardly directed sucrose gradient. (A) Representative time course data for negative control (0.5% DMSO vehicle alone), 0.3 mM HgCl₂ (positive control), and indicated compounds (each 50 μM). Cells and ghosts were incubated with test compounds for ∼15 minutes before measurements. (B) Relative osmotic water permeability (S.E., n = 4). *P < 0.05 compared with control.

Fig. 7.

Osmotic water permeability in plasma membrane vesicles from CHO cells. Osmotic water permeability was measured from the time course of scattered-light intensity in response to a 250 mM inwardly directed sucrose gradient. (A) Representative time course data for negative control (0.5% DMSO vehicle alone) and 0.3 mM HgCl₂ in vesicles from nontransfected CHO cells expressing AQP1- and AQP4-M23. (B) Representative time course data for indicated compounds (each 50 μM). Vesicles were incubated with test compounds for ∼15 minutes before measurements. (C) Relative osmotic water permeability (S.E., n = 4). *P < 0.05 compared with control.