Inhibition of ATP hydrolysis restores airway surface liquid production in cystic fibrosis airway epithelia

Airway surface dehydration is a pathological feature of cystic fibrosis (CF) lung disease. 20 CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cyclic 21 AMP-regulated Cl- channel controlled in part by the adenosine A2B receptor. An alternative, 22 CFTR-independent mechanism of fluid secretion is regulated by ATP, via the P2Y2 receptor 23 (P2Y2R) that activates Ca2+-regulated Cl- channels (CaCC/TMEM16) and inhibits Na+ 24 absorption. However, due to rapid ATP hydrolysis, steady-state ATP levels in CF airway surface 25 liquid (ASL) are inadequate to maintain P2Y2R-mediated fluid secretion. Therefore, inhibiting 26 airway epithelial ecto-ATPases to increase ASL ATP levels constitutes a strategy to restore 27 airway surface hydration in CF. Using [γ32P]ATP as radiotracer, we assessed the effect of a 28 series of ATPase inhibitory compounds on the stability of physiologically occurring ATP 29 concentrations. We identified the polyoxometalate [Co4(H2O)2(PW9O34)2]10- (POM-5) as the 30 most potent and effective ecto-ATPase inhibitor in CF airway epithelial cells. POM-5 caused 31 long-lasting inhibition of ATP hydrolysis in airway epithelia, which was reversible upon removal 32 of the inhibitor. Importantly, POM-5 markedly enhanced steady-state levels of released ATP, 33 promoting increased ASL volume in CF cell surfaces. These results provide proof-of-concept for 34 ecto-ATPase inhibitors as therapeutic agents to restore hydration of CF airway surfaces. As a test 35 of this notion, cell-free sputum supernatants from CF subjects were studied and found to have 36 abnormally elevated ATPase activity, which was markedly inhibited by POM-5.