Automated cell-based assay for screening of aquaporin inhibitors

Aquaporins form water channels that play major roles in a variety of physiological processes so that altered expression or function may underlie pathological conditions. In order to identify compounds that modulate aquaporin function, we have implemented a functional assay based on rapid measurement of osmotically induced cell volume changes to screen several libraries of diverse drugs. The time course of fluorescence changes in calcein-loaded cells was analyzed during an osmotic challenge using a 96-multiwell fluorescence plate reader. This system was validated using astrocyte primary cultures and fibroblasts that strongly express endogenous AQP4 and AQP1 proteins, respectively, as well as AQP4-transfected cells. We screened 3575 compounds, including 418 FDA-approved and commercially available drugs, for their effect on AQP-mediated water transport. Primary screening yielded 10 compounds that affected water transport activity in both astrocytes and AQP4-transfected cells and 42 compounds that altered cell volume regulation in astrocytes. Selected drugs were then analyzed on AQP1-expressing erythrocytes and AQP4-expressing membrane vesicles by stopped-flow light scattering. Four molecules of the National Cancer Institute's chemical library (NSC164914, NSC670229, NSC168597, NSC301460) were identified that differentially affected both AQP4 and AQP1 mediated water transport, with EC₅₀ values between 20 and 50 μM. This fluorescence microplate reader-based assay may, thus, provide a platform for highthroughput screening which, when coupled to a secondary evaluation to confirm target specificity, should allow discovery of AQP-specific compounds for novel therapeutic strategies in the treatment of water balance disorders.