Aim: 

The importance of aquaporin channels in both physiology and pathophysiology is well established. A number of studies have suggested that their pharmacological modulation may give further insights into understanding the molecular mechanism of water transport and provide novel intervention opportunities in a variety of human disorders. Here, we used a high-throughput screening to identify chemicals compounds that can modulate aquaporins function selectively and with high affinity.

Methods: 

We set up a functional assay based on measurement of osmotically-induced cell volume changes by using calcein quenching method. Time courses of fluorescence were analyzed using a multireader fluorimeter suitable for microplates records. A collection of 3500 small drug-like compounds was screened using a cell-based fluorescence assay. We used mouse astrocyte primary culture, 2497-fibroblast cell line that strongly express endogenous AQP4 and AQP1 proteins respectively and TNC-1 cells transfected with AQP4.

Results: 

Primary screening yielded about 10 chemical compounds able to affect water transport activity and/or cell volume regulation in both astrocytes and AQP4-TNC cells. The selected drugs were then screened on erythrocytes and membrane vesicles obtained from AQP4-transfected TNC1 cells by using a stopped-flow light scattering method to eliminate false positive hits due to calcein fluorescence. Six molecules were identified and interestingly two of them containing metals have an inhibitory effect on both AQP4 and AQP1 mediated water transport with an IC₅₀ between 20 and 50 mM.

Conclusion: 

We characterize here the sensitivity of a fluorescence microplate reader-based assay and demonstrate that it could represent a useful approach to discover potential pharmacological modulators of water channel activity suitable for a novel therapeutic strategy.