Identification of a chemical probe for NAADP by virtual screening

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.

References

  1. 1

    Lee, H.C. Nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated calcium signaling. J. Biol. Chem. 280, 33693–33696 (2005).

    CAS  Article  Google Scholar 

  2. 2

    Galione, A., Parrington, J. & Dowden, J. The NAADP receptor: commentary on Billington et al. Br. J. Pharmacol. 142, 1203–1207 (2004).

    CAS  Article  Google Scholar 

  3. 3

    Lee, H.C. & Aarhus, R. A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cyclic ADP-ribose. J. Biol. Chem. 270, 2152–2157 (1995).

    CAS  Article  Google Scholar 

  4. 4

    Giniatullin, R., Nistri, A. & Yakel, J.L. Desensitization of nicotinic ACh receptors: shaping cholinergic signaling. Trends Pharmacol. Sci. 28, 371–378 (2005).

    CAS  Google Scholar 

  5. 5

    Cancela, J.M., Churchill, G.C. & Galione, A. Coordination of agonist-induced Ca2+-signalling patterns by NAADP in pancreatic acinar cells. Nature 398, 74–76 (1999).

    CAS  Article  Google Scholar 

  6. 6

    Masgrau, R., Churchill, G.C., Morgan, A.J., Ashcroft, S.J. & Galione, A. NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells. Curr. Biol. 13, 247–251 (2003).

    CAS  Article  Google Scholar 

  7. 7

    Patel, S. NAADP-induced Ca2+ release–a new signalling pathway. Biol. Cell 96, 19–28 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Churchill, G.C. et al. NAADP mobilizes Ca2+ from reserve granules, lysosome-related organelles, in sea urchin eggs. Cell 111, 703–708 (2002).

    CAS  Article  Google Scholar 

  9. 9

    Yamasaki, M. et al. Organelle selection determines agonist-specific Ca2+ signals in pancreatic acinar and beta cells. J. Biol. Chem. 279, 7234–7240 (2004).

    CAS  Article  Google Scholar 

  10. 10

    Gerasimenko, J.V., Sherwood, M., Tepikin, A.V., Petersen, O.H. & Gerasimenko, O.V. NAADP, cADPR and IP3 all release Ca2+ from the endoplasmic reticulum and an acidic store in the secretory granule area. J. Cell Sci. 119, 226–238 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Galione, A. & Petersen, O.H. The NAADP receptor: new receptors or new regulation? Mol. Interv. 5, 73–79 (2005).

    CAS  Article  Google Scholar 

  12. 12

    Mitchell, K.J., Lai, F.A. & Rutter, G.A. Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6). J. Biol. Chem. 278, 11057–11064 (2003).

    CAS  Article  Google Scholar 

  13. 13

    Genazzani, A.A., Empson, R.M. & Galione, A. Unique inactivation properties of NAADP-sensitive Ca2+ release. J. Biol. Chem. 271, 11599–11602 (1996).

    CAS  Article  Google Scholar 

  14. 14

    Morgan, A.J. et al. Methods in cyclic ADP-ribose and NAADP research. in Methods in Calcium Signalling (ed. Putney, J.W.J.) 265–333 (CRC Press, Boca Raton, Florida, USA, 2006).

    Google Scholar 

  15. 15

    Brailoiu, E. et al. Nicotinic acid adenine dinucleotide phosphate potentiates neurite outgrowth. J. Biol. Chem. 280, 5646–5650 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Billington, R.A. et al. Production and characterization of reduced NAADP (nicotinic acid-adenine dinucleotide phosphate). Biochem. J. 378, 275–280 (2004).

    CAS  Article  Google Scholar 

  17. 17

    Billington, R.A., Tron, G.C., Reichenbach, S., Sorba, G. & Genazzani, A.A. Role of the nicotinic acid group in NAADP receptor selectivity. Cell Calcium 37, 81–86 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Lee, H.C. & Aarhus, R. Structural determinants of nicotinic acid adenine dinucleotide phosphate important for its calcium-mobilizing activity. J. Biol. Chem. 272, 20378–20383 (1997).

    CAS  Article  Google Scholar 

  19. 19

    Lee, H.C. & Aarhus, R. Fluorescent analogs of NAADP with calcium mobilizing activity. Biochim. Biophys. Acta 1425, 263–271 (1998).

    CAS  Article  Google Scholar 

  20. 20

    Dowden, J. et al. Cell-permeant small-molecule modulators of NAADP-mediated Ca2+ release. Chem. Biol. 13, 659–665 (2006).

    CAS  Article  Google Scholar 

  21. 21

    Lipinski, C.A., Lombardo, F., Dominy, B.W. & Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 23, 3–25 (1997).

    CAS  Article  Google Scholar 

  22. 22

    Billington, R.A., Bak, J., Martinez-Coscolla, A., Debidda, M. & Genazzani, A.A. Triazine dyes are agonists of the NAADP receptor. Br. J. Pharmacol. 142, 1241–1246 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Billington, R.A. & Genazzani, A.A. PPADS is a reversible competitive antagonist of the NAADP receptor. Cell Calcium 41, 505–511 (2007).

    CAS  Article  Google Scholar 

  24. 24

    Jorgensen, W.L. The many roles of computation in drug discovery. Science 303, 1813–1818 (2004).

    CAS  Article  Google Scholar 

  25. 25

    Oprea, T.I. & Matter, H. Integrating virtual screening in lead discovery. Curr. Opin. Chem. Biol. 8, 349–358 (2004).

    CAS  Article  Google Scholar 

  26. 26

    Shoichet, B.K. Virtual screening of chemical libraries. Nature 432, 862–865 (2004).

    CAS  Article  Google Scholar 

  27. 27

    Drews, J. Drug discovery: a historical perspective. Science 287, 1960–1964 (2000).

    CAS  Article  Google Scholar 

  28. 28

    Klebe, G. Virtual ligand screening: strategies, perspectives and limitations. Drug Discov. Today 11, 580–594 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Irwin, J.J. & Shoichet, B.K. ZINC–a free database of commercially available compounds for virtual screening. J. Chem. Inf. Model. 45, 177–182 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Rush, T.S. III, Grant, J.A., Mosyak, L. & Nicholls, A. A shape-based 3-D scaffold hopping method and its application to a bacterial protein-protein interaction. J. Med. Chem. 48, 1489–1495 (2005).

    CAS  Article  Google Scholar 

  31. 31

    Nicholls, A., MacCuish, N.E. & MacCuish, J.D. Variable selection and model validation of 2D and 3D molecular descriptors. J. Comput. Aided Mol. Des. 18, 451–474 (2004).

    CAS  Article  Google Scholar 

  32. 32

    Galione, A. & Ruas, M. NAADP receptors. Cell Calcium 38, 273–280 (2005).

    CAS  Article  Google Scholar 

  33. 33

    Boström, J., Berggren, K., Elebring, T., Greasleya, P.J. & Wilstermanna, M. Scaffold hopping, synthesis and structure–activity relationships of 5,6-diaryl-pyrazine-2-amide derivatives: a novel series of CB1 receptor antagonists. Bioorg. Med. Chem. 15, 4077–4084 (2007).

    Article  Google Scholar 

  34. 34

    Hawkins, P.C.D., Skillman, A.G. & Nicholls, A. Comparison of shape-matching and docking as virtual screening tools. J. Med. Chem. 50, 74–82 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Jenkins, J.L., Glick, M. & Davies, J.W. A 3D similarity method for scaffold hopping from known drugs or natural ligands to new chemotypes. J. Med. Chem. 47, 6144–6159 (2004).

    CAS  Article  Google Scholar 

  36. 36

    Boström, J., Greenwood, J.R. & Gottfries, J. Assessing the performance of OMEGA with respect to retrieving bioactive conformations. J. Mol. Graph. Model. 21, 449–462 (2003).

    Article  Google Scholar 

  37. 37

    Grant, J.A., Gallardo, M.A. & Pickup, B.T. A fast method of molecular shape comaprison: a simple application of a Gaussian description of molecular shape. J. Comput. Chem. 17, 1653–1666 (1996).

    CAS  Article  Google Scholar 

  38. 38

    Doman, T.N. et al. Molecular docking and high-throughput screening for novel inhibitors of protein tyrosine phosphatase-1B. J. Med. Chem. 45, 2213–2221 (2002).

    CAS  Article  Google Scholar 

  39. 39

    Aarhus, R. et al. Activation and inactivation of Ca2+ release by NAADP+. J. Biol. Chem. 271, 8513–8516 (1996).

    CAS  Article  Google Scholar 

  40. 40

    Giepmans, B.N., Adams, S.R., Ellisman, M.H. & Tsien, R.Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    CAS  Article  Google Scholar 

  41. 41

    Johnson, J.D. & Misler, S. Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signalling in human beta cells. Proc. Natl. Acad. Sci. USA 99, 14566–14571 (2002).

    CAS  Article  Google Scholar 

  42. 42

    Arredouani, A., Henquin, J.C. & Gilon, P. Contribution of the endoplasmic reticulum to the glucose-induced Ca2+ response in mouse pancreatic islets. Am. J. Physiol. Endocrinol. Metab. 282, E982–E991 (2002).

    CAS  Article  Google Scholar 

  43. 43

    Tan, D.S. Diversity-orientated synthesis: exploring the intersections between biology and chemistry. Nat. Chem. Biol. 1, 74–84 (2005).

    CAS  Article  Google Scholar 

  44. 44

    Ashcroft, F.M., Harrison, D.E. & Ashcroft, S.J. Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells. Nature 312, 446–448 (1984).

    CAS  Article  Google Scholar 

  45. 45

    Seghers, V., Nakazaki, M., DeMayo, F., Aguilar-Bryan, L. & Bryan, J. Sur1 knockout mice. A model for KATP channel-independent regulation of insulin secretion. J. Biol. Chem. 275, 9270–9277 (2000).

    CAS  Article  Google Scholar 

  46. 46

    Islam, M.S., Larsson, O. & Berggren, P.O. Cyclic ADP-ribose in beta cells. Science 262, 584–586 (1993).

    CAS  Article  Google Scholar 

  47. 47

    Takasawa, S., Nata, K., Yonekura, H. & Okamoto, H. Cyclic ADP-ribose in insulin secretion from pancreatic beta cells. Science 259, 370–373 (1993).

    CAS  Article  Google Scholar 

  48. 48

    Parkesh, R. et al. Cell-permeant NAADP: a novel chemical tool enabling the study of Ca2+ signalling in intact cells. Cell Calcium 43, 531–538 (2007).

    Article  Google Scholar 

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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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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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Supplementary Figures 1 and 2, Supplementary Scheme 1, Supplementary Table 1 and Supplementary Methods (PDF 1560 kb)

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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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