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.
Highlights
The aquaglyceroporins are a subfamily of aquaporins that conduct both water and glycerol
Chinese hamster ovary (CHO) cells expressing mAQP3 under the control of a tetracycline promoter were grown in 96-well plates for identifying AQP3 inhibitors in a calcein quenching– based assay of cell water permeability [34]
We describe the identification of novel mAQP3 and mAQP7 inhibitors
Summary
Chinese hamster ovary (CHO) cells expressing mAQP3 under the control of a tetracycline promoter were grown in 96-well plates for identifying AQP3 inhibitors in a calcein quenching– based assay of cell water permeability [34]. Hits could broadly be categorized into the single hit BTB09519 and a group of substances described by a urea-containing linker (DFP00176, GK00877, or SEW00832) or acetamide linker (BTB14129); a left-hand 2-nitrothiophene, ethyl-benzoate, or chlorobenzene group; and a more variable right-hand side, as intuitively depicted (Fig. 1). These substances shared structural elements that are similar to the previously identified benzothiadiazole urea– containing AQP9 inhibitors RF03176 and HTS13772 [34, 35]. No clear potency differences were observed between the inhibition of mAQP3 and hAQP3
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