Discovery and Characterization of Novel Selective NKCC1 Inhibitors for Down Syndrome and Brain Disorders with Depolarizing GABAergic Transmission

Proper GABAergic transmission through Cl-permeable GABAA receptors is fundamental for physiological brain development and function. Indeed, defective GABAergic signaling -due to a high ratio of expression of the Cl importer NKCC1 and Cl exporter KCC2- has been implicated in several neurodevelopmental disorders (e.g., Down syndrome, DS). Interestingly, NKCC1 inhibition by the FDAapproved diuretic bumetanide reverts cognitive deficits in the TS65Dn mouse models of DS and core symptoms in a number of models of other brain disorders. However, the required chronic treatment with bumetanide is burdened by its diuretic side effects caused by the antagonism of the kidney Cl- importer NKCC2, which leads to hypokalemia and jeopardizes drug compliance. Crucially, these issues would be solved by selective NKCC1 inhibitors, thus devoid of the diuretic effect. Starting from bumetanide’s structure, we applied a computational ligand-based approach to design new molecular entities that we tested in vitro for their capacity to selectively block NKCC1. Among the 3 newly-identified and highly promising NKCC1 inhibitors, one showed excellent solubility and metabolic stability in vitro. Moreover, analysis of WT and Ts65Dn mice systemically treated with this NKCC1 inhibitor revealed no diuretic effect. Finally, chronic treatment with our novel, selective NKCC1 inhibitor was able to rescue cognitive deficits in Ts65Dn mice in four different memory tasks, with no major signs of toxicity. Thus, our selective NKCC1 inhibitor devoid of the diuretic effect could represent a suitable and solid therapeutic strategy for the treatment of Down syndrome and all the brain disorders with depolarizing GABAergic transmission.