Identification of a chemical probe for NAADP by virtual screening

Nature Chemical Biology volume 5, pages220–226(2009)Cite this article

Abstract

Research into the biological role of the Ca2+-releasing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate) has been hampered by a lack of chemical probes. To find new chemical probes for exploring NAADP signaling, we turned to virtual screening, which can evaluate millions of molecules rapidly and inexpensively. We used NAADP as the query ligand to screen the chemical library ZINC for compounds with similar three-dimensional shape and electrostatic properties. We tested the top-ranking hits in a sea urchin egg bioassay and found that one hit, Ned-19, blocks NAADP signaling at nanomolar concentrations. In intact cells, Ned-19 blocked NAADP signaling and fluorescently labeled NAADP receptors. Moreover, we show the utility of Ned-19 as a chemical probe by using it to demonstrate that NAADP is a key causal link between glucose sensing and Ca2+ increases in mouse pancreatic beta cells.

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Figure 1: Strategy and results of a ligand-based virtual screen for drug-like molecules with NAADP-like activity.
Figure 2: Certain virtual screening hits have biological activity.
Figure 3: The virtual screening hit Ned-19 antagonizes Ca2+ signaling in intact cells.
Figure 4: The virtual screening hit Ned-19 is fluorescent and labels receptors in intact cells.
Figure 5: The virtual screening hit Ned-19 reveals that glucose-induced Ca2+ increases require NAADP signaling.

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Acknowledgements

Our research was supported by a grant from the Biotechnology and Biological Sciences Research Council (grant number BB/D012694/1). We thank P. Hawkins (OpenEye Scientific Software) for advice with virtual screening, H.-C. Lee (University of Hong Kong) for providing ADP-ribosyl cyclase and C. Garnham (Oxford University) for help with the plate reader.

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Author notes

  1. Edmund Naylor, Abdelilah Arredouani and Sridhar R Vasudevan: These authors contributed equally to this work.

Affiliations

  1. Department of Pharmacology, University of Oxford, Oxford, UK

    Edmund Naylor, Abdelilah Arredouani, Sridhar R Vasudevan, Alexander M Lewis, Raman Parkesh, Daniel Rosen, Justyn M Thomas, Antony Galione & Grant C Churchill

  2. Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan

    Akiko Mizote & Minoru Izumi

  3. School of Chemistry, University of Southampton, Highfield, Southampton, UK

    A Ganesan

Authors
  1. Edmund Naylor
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  2. Abdelilah Arredouani
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  10. A Ganesan
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  11. Antony Galione
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  12. Grant C Churchill
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Contributions

E.N., S.R.V. and R.P., initial virtual screening; E.N., initial biological testing; A.A. and A. Galione, electrophysiology and calcium imaging of beta cells; G.C.C., calcium imaging of urchin eggs; and S.R.V., detailed virtual screening. A.M.L., binding, plate reading and fluorimetry; G.C.C. and J.M.T, and fluorimetry; A.M., M.I. and A. Ganesan, chemical synthesis and characterization; and D.R., diastereomer binding and fluorimetry. G.C.C., principal investigator, designed and planned the project, wrote and handled the manuscript.

Corresponding author

Correspondence to Grant C Churchill.

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Competing interests

The authors are going to attempt to patent the diastereomers of Ned-19.

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Naylor, E., Arredouani, A., Vasudevan, S. et al. Identification of a chemical probe for NAADP by virtual screening. Nat Chem Biol 5, 220–226 (2009). https://doi.org/10.1038/nchembio.150

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