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

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An Erratum to this article was published on 15 April 2011

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 [γ 32P]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 [γ 32P]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 ± 2 μM). 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 ± 3 μM), having negligible effect on K M values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK.

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Acknowledgments

We thank Dr. Scott Randell for providing primary cultures of HBE cells. We are indebted to Lisa Brown for editorial assistance with the manuscript. This work was supported, in whole or in part, by National Institutes of Health Grant P01-HL034322 (ERL, CH) and Cystic Fibrosis Foundation Grant CFF-SEMINA08FO (LS-V). GM was supported by the Department of Science and Technology (DST), India.

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Authors and Affiliations

  1. Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina School of Medicine, 7017 Thurston-Bowles Building, CB 7248, Chapel Hill, NC, 27599-7248, USA

    Lucia Semianrio-Vidal, Catharina van Hesuden & Eduardo Rodolfo Lazarowski

  2. Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, India

    Govindasamy Mugesh

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  1. Lucia Semianrio-Vidal
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  2. Catharina van Hesuden
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  3. Govindasamy Mugesh
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  4. Eduardo Rodolfo Lazarowski
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Correspondence to Eduardo Rodolfo Lazarowski.

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An erratum to this article can be found at http://dx.doi.org/10.1007/s11302-011-9230-2

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Semianrio-Vidal, L., van Hesuden, C., Mugesh, G. et al. Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase. Purinergic Signalling 6, 383–391 (2010). https://doi.org/10.1007/s11302-010-9203-x

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