Identification and characterization of potent and selective aquaporin-3 and aquaporin-7 inhibitors

Abstract

The aquaglyceroporins are a subfamily of aquaporins that conduct both water and glycerol. Aquaporin-3 (AQP3) has an important physiological function in renal water reabsorption, and AQP3-mediated hydrogen peroxide (H₂O₂) permeability can enhance cytokine signaling in several cell types. The related aquaglyceroporin AQP7 is required for dendritic cell chemokine responses and antigen uptake. Selective small-molecule inhibitors are desirable tools for investigating the biological and pathological roles of these and other AQP isoforms. Here, using a calcein fluorescence quenching assay, we screened a library of 7360 drug-like small molecules for inhibition of mouse AQP3 water permeability. Hit confirmation and expansion with commercially available substances identified the ortho-chloride-containing compound DFP00173, which inhibited mouse and human AQP3 with an IC₅₀ of ∼0.1-0.4 μm but had low efficacy toward mouse AQP7 and AQP9. Surprisingly, inhibitor specificity testing revealed that the methylurea-linked compound Z433927330, a partial AQP3 inhibitor (IC₅₀, ∼0.7-0.9 μm), is a potent and efficacious inhibitor of mouse AQP7 water permeability (IC₅₀, ∼0.2 μm). Stopped-flow light scattering measurements confirmed that DFP00173 and Z433927330 inhibit AQP3 glycerol permeability in human erythrocytes. Moreover, DFP00173, Z433927330, and the previously identified AQP9 inhibitor RF03176 blocked aquaglyceroporin H₂O₂ permeability. Molecular docking to AQP3, AQP7, and AQP9 homology models suggested interactions between these inhibitors and aquaglyceroporins at similar binding sites. DFP00173 and Z433927330 constitute selective and potent AQP3 and AQP7 inhibitors, respectively, and contribute to a set of isoform-specific aquaglyceroporin inhibitors that will facilitate the evaluation of these AQP isoforms as drug targets.

Keywords: CHO cell assay; HyPer; aquaporin; aquaporin inhibitor; calcein; glycerol; hydrogen peroxide; inhibitor; molecular docking; residual pore.

Figure 1.

Dose–response curves for the ten most potent screening hits as well as for the positive control HTS06792. Shown is t_½ of cell shrinking, indicating that water permeability was measured in calcein-loaded, mAQP3-expressing CHO cells. Screening hits were repurchased before analysis to confirm hit identity. Supplier product codes are indicated above each substance. Compounds MWP00821 and S04288 were not sufficiently soluble in DMSO and were thus tested at lower concentrations than the other compounds. AQP3 inhibition could be confirmed for five hit compounds. In addition, JM00015 showed AQP3 inhibition at the highest tested concentration and at lower efficacy than expected.

Figure 2.

Potency and structure–activity relationship analysis of mAQP3 and hAQP3 inhibition, respectively, by commercially available hit analogues. Shown is t_½ of cell shrinking, indicating that water permeability was measured in calcein-loaded, m/hAQP3-expressing CHO cells. No clear potency differences between mAQP3 and hAQP3 inhibition were observed. Error bars indicate standard deviation.

Figure 3.

AQP isoform specificity of three selected compounds. A–C, cell water permeability was measured in calcein-loaded recombinant CHO cell lines. The structurally similar compounds 9016645 (A) and Z433927330 (B) show reversed isoform specificity. DFP00173 (C) specifically inhibits AQP3 over the homologous AQP isoforms AQP7 and AQP9. 9016645: IC₅₀, ∼6 μm (mAQP3); IC₅₀, N.D. (mAQP7); IC₅₀, N.D. (mAQP9). Z433927330: IC₅₀, ∼0.2 μm (mAQP7); IC₅₀, ∼0.7 μm (mAQP3); IC₅₀, ∼1.1 μm (mAQP9). DFP00173: IC₅₀, ∼0.1 μm (mAQP3); IC₅₀, N.D. (mAQP7); IC₅₀, N.D. (mAQP9). The AQP isoform was identified as a significant source for variation in two-way ANOVA for all three substances at p < 0.0001; n = 3. D, cell proliferation and viability of CHO cells are not reduced after 48 h of incubation with AQP inhibitors compared with solvent (DMSO). 1, DFP00173; 2, Z433927330; 3, RF03176. Calcein retention was not affected at 2.5 μm. Exposure to 25 μm of either DFP00173 or Z433927330 resulted in increased fluorescence (p < 0.001; n = 4 for inhibitors and 8 for DMSO). Error bars indicate standard deviation. AU, arbitrary units.

Figure 4.

Shown are H₂O₂ permeability (top row) and water permeability (bottom row) in H₂O₂ sensor (HyPer-3)–expressing CHO cell lines. Expression of mAQP3, mAPQ7, and mAPQ9 conferred a faster fluorescence increase in response to H₂O₂ addition (150 μm), compared with control cells (no AQP). Addition of cognate inhibitors resulted in a slower fluorescence increase in AQP-expressing cell lines after H₂O₂ addition, whereas no clear effects on control cells (no AQP) were observed. Similarly, AQP inhibitors reduced water permeability in AQP-expressing cell lines only (cell shrinking in response to sucrose addition 3.6 s into each read). The same HyPer-3–expressing cell lines were loaded with calcein before water permeability measurements. We note that the calcein fluorescence intensity is about 10-fold higher than HyPer-3 fluorescence under the conditions used. HyPer-3 did not seem to interfere with water permeability measurements. Furthermore, we noted a small baseline fluorescence decrease induced by DFP00173 and RF03176 treatment that was present before H₂O₂ addition. The reason for this effect is unknown. Means of four recordings are shown.

Figure 5.

Inhibition of glycerol permeability. A, addition of extracellular glycerol causes a rapid scattered light intensity (SLI) increase, indicative of water exit, mainly through hAQP1 and hAPQ3, followed by a slower scattered light intensity decrease, indicative of swelling induced by the osmotic influx of water, following glycerol entry mainly through AQP3. Representative example traces show clear inhibition of erythrocyte water and glycerol permeability by addition of 25 μm DFP00173 compared with solvent (1% DMSO). B, relative potency of erythrocyte glycerol permeability inhibition, measured as t_½ of the scattered light intensity decrease. Apparent IC₅₀ values were ∼0.2 μm (DFP00173) and ∼0.6 μm (Z433927330). Substance was identified as a significant source for variation in two-way ANOVA at p < 0.0001; n = 6. C, example traces of glycerol permeability in calcein-loaded CHO-mAQP3 cells in an isotonic shrinking assay. DFP00173-treated (25 μm) and solvent control (1% DMSO) cell traces of relative fluorescence intensity are shown. Cells were preincubated in 500 mm glycerol buffer before addition of 500 mm membrane-impermeable sucrose. Fluorescence quenching indicates cellular glycerol efflux along its gradient. D, dose–response curves of glycerol permeability inhibition in CHO cells expressing mAQP3 and mAQP7, respectively. Fluorescence quenching kinetics expressed as t_½ were measured. Apparent IC₅₀ values were ∼0.7 μm for DFP00173 (mAQP3), N.D. for DFP00173 (mAQP7), ∼11.5 μm for Z433927330 (mAQP3), and ∼2.6 μm for Z433927330 (mAQP7). n = 3. AU, arbitrary units. Error bars in B and D indicate standard deviation.

Figure 6.

Representative AQP-inhibitor binding sites. A–C, the calculated diameter of the AQP pore is shown in transparent surface view, colored from blue (narrowest) to red (widest). Docked ligands and select amino acids are shown in stick representation. Hydrogen bonds form between the inhibitor urea linker and backbone carbonyls of each AQP isoform. Asparagines of the NPA boxes form interactions between AQP3 and DFP00173 (A) and between AQP7 and Z433927330 (B). Phe-180 is unique to AQP9 and may form a positive edge interaction with an RF03176 nitrogen lone pair, thereby stabilizing a conformation that constricts the entrance to the pore (C).