Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins

https://doi.org/10.1016/j.mimet.2015.11.012Get rights and content

Highlights

  • Purified respiratory Type II NADH dehydrogenase (NDH-2) was lipid-reconstituted.

  • Detergent-solubilised and lipid-reconstituted NDH-2 was screened for inhibitors.

  • Inhibitors of lipid-reconstituted NDH-2 were more potent and less lipophilic.

  • Lipid-reconstituted respiratory membrane-protein drug-screening may be advantageous.

Abstract

This study compared detergent-solubilised (soluble) and lipid-reconstituted (proteoliposome) protein to establish a high-throughput method for identifying membrane protein inhibitors. We identified inhibitors of the membrane-bound type II NADH dehydrogenase with lower lipophilicity and better potency, suggesting proteoliposome systems may be advantageous over detergent-solubilised systems for respiratory membrane proteins.

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Author contributions

E.D., G.M.C. and A.H. designed the research, E.D. performed the research, E.D., G.M.C. and A.H. analysed the data and E.D., G.M.C. and A.H. wrote the paper.

Declaration of conflicting interests

The authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Acknowledgements and funding

This work was supported by the Health Research Council of New Zealand and the Maurice Wilkins Centre for Molecular Biodiscovery. E. Dunn was funded by research scholarships from the University of Otago and The Todd Foundation New Zealand. We thank Ivan Summit for equipment use, and Dr. Mattie Timmer and Dr. Bridget Stocker for the Quinolinequinone Library.

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    The table contains the library; compound name, number of H-bond donors/acceptors, molecular weight, lipophilicity (logP) and degree of inhibition/stimulation for DS and LR protein (Table 1). Type II NADH dehydrogenase (NDH-2) was purified and reconstituted into proteoliposomes as described by Dunn et al. [2,3]. Detergent-solubilised (DS) NDH-2 or lipid-reconstituted (LR) NDH-2 were screened by end point 96-well assay (340 nm, Varioskan® Flash Thermo Scientific, in the presence of 100 μM menadione and 20 μM test compound.

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