Central nervous system (CNS) oedema is a life-threatening condition that affects millions of people each year and for which there is no cure. It results from abnormal water accumulation in the brain, often caused by trauma, stroke, or infection. Current treatments focus solely on managing symptoms, highlighting the urgent need for effective pharmacological therapies to improve clinical outcomes. Aquaporin-4 (AQP4), a water channel highly expressed in astrocytic endfeet, plays a key role in dysregulated fluid homeostasis after CNS injury, making it a promising therapeutic target. The rapid cellular swelling observed in CNS oedema is partly mediated by the upregulation and membrane localization of AQP4 in astrocytes, which involves direct interaction of calmodulin with AQP4. In a rat spinal cord injury model, our research group showed that inhibiting AQP4 relocalization to the plasma membrane by targeting this interaction effectively reduced spinal cord oedema and improved recovery (Kitchen et al., 2020). Therefore, our research proposes an innovative shift by developing small molecules that specifically inhibit AQP4 trafficking to the membrane or directly block its water channel function. By preventing or reversing oedema, we aim to reduce secondary injury cascades to promote recovery after CNS injury.

We employ an integrated discovery pipeline to identify small-molecule modulators of AQP4 function. Novel compounds are designed using computer-aided drug design and subsequently synthesized, purified, and fully characterized in-house. To target AQP4 trafficking, candidate inhibitors are screened in vitro for their ability to disrupt the interaction between the AQP4 and calmodulin, using flow-induced dispersion analysis. Compounds are evaluated in cell-based oedema models for their capacity to reduce AQP4 abundance at the plasma membrane under conditions that promote AQP4 relocalization, namely hypotonicity- and hypoxia-induced cell swelling. In parallel, we pursue AQP4 direct blockers. These compounds are screened in vitro for binding to AQP4 using a tryptophan quenching assay, with functional validation in cell-based systems. Their ability to inhibit water transport is tested by measuring water efflux in primary human astrocytes and AQP4-overexpressing MDCK cells under hypertonic conditions using a calcein quenching assay. Together with toxicity, off-target, and stability assessments, these functional assays inform medicinal chemistry optimisation toward the development of lead compounds.

Using this multi-layered approach, we have recently identified several promising hit molecules. Lead compounds will ultimately be advanced to in vivo testing in a CNS injury model and will build a framework for pharmacological intervention for the millions of people affected by CNS oedema worldwide every year.