Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase

Abstract

Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase and adenyl kinase activities. Addition of radiotracer [γ³²P]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of [³²P]-inorganic phosphate (³²Pi). Inclusion of UDP in the incubation medium resulted in conversion of [γ³²P]ATP to [³²P]UTP, while inclusion of AMP resulted in conversion of [γ ³²P]ATP to [³²P]ADP. Ebselen markedly reduced [³²P]UTP formation but displayed negligible effect on ³²Pi or [³²P]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of ATP (IC₅₀=6.9±2μM). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549 cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the V_max of the reaction (K_i=7.6±3μM), having negligible effect on K_M values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK.