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Identification of a chemical probe for NAADP by virtual screening

Nature Chemical Biology volume 5, pages 220226 (2009) | Download Citation

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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.

  • Compound

    Nicotinic acid adenine dinucleotide phosphate

  • Compound

    Bafilomycin A1

  • Compound

    Thapsigargin

  • Compound

    Glycylphenylalanine 2-naphthylamide

  • Compound

    4-[(2E)-2-[4-Formyl-6-methyl-5-oxo-3-(phosphonooxymethyl)pyridin-2-ylidene]hydrazinyl]benzene-1,3-disulfonic acid

  • Compound

    1-(3-((4-(2-Fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid

  • Compound

    (1R,3S)-1-(3-((4-(2-Fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid

  • Compound

    (1S,3S)-1-(3-((4-(2-Fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid

  • Compound

    N,N′-(2,2-Dimethylpropane-1,3-diyl)bis(2-(5-methylfuran-2-yl)quinoline-4-carboxamide)

  • Compound

    (E)-N′-(3-Phenoxybenzylidene)-2-(5-phenyl-4-p-tolyl-4H-1,2,4-triazol-3-ylthio)acetohydrazide

  • Compound

    2-(N-(4-Fluorophenyl)phenylsulfonamido)-N-(4-(N-naphthalen-1-ylsulfamoyl)phenyl)acetamide

  • Compound

    N-(4-(Isopropyl(phenyl)amino)phenyl)-4-(N-(2-methoxyphenyl)-N-methylsulfamoyl)benzamide

  • Compound

    N-(2-Benzoyl-4-chlorophenyl)-2-(3-(3,5-dimethylphenoxy)-4-oxo-4H-chromen-7-yloxy)acetamide

  • Compound

    (Z)-2-(5-(4-Chlorophenyl)-4-p-tolyl-4H-1,2,4-triazol-3-ylthio)-N′-(3-phenoxybenzylidene)acetohydrazide

  • Compound

    (E)-4-(2-(2-Benzoyl-4-chlorophenyl)hydrazono)-5-methylene-2-(4-(3-nitrophenyl)thiazol-2-yl)pyrazolidin-3-one

  • Compound

    2-(9H-Fluoren-3-yl)-2-oxoethyl 3-(3,4-dihydroquinolin-1(2H)-ylsulfonyl)-4-methoxybenzoate

  • Compound

    2-(Biphenyl-2-yloxy)-1-(4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazin-1-yl)ethanone

  • Compound

    3-(2-(2-((Z)-((8R,9R,10R,13R,14S,17S)-17-Hydroxy-10,13,17-trimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-ylidene)aminooxy)acetamido)ethyl)-5-methoxy-1H-indole-2-carboxylate

  • Compound

    1,1′-(5S,5′S)-3,3′-(1,4-Phenylene)bis(5-(2,3-dimethoxyphenyl)-1H-pyrazole-3,1(4H,5H)-diyl)dipropan-1-one

  • Compound

    2-Oxopropane-1,3-diyl bis(3-phenoxybenzoate)

  • Compound

    (E)-N-Benzyl-N-(4-(2-(3-phenoxybenzylidene)hydrazinecarbonyl)phenyl)methanesulfonamide

  • Compound

    ((1S,9aR)-Octahydro-1H-quinolizin-1-yl)methyl 4-((R)-2-(1,3-dioxoisoindolin-2-yl)propanamido)benzoate

  • Compound

    2-(Diethylamino)ethyl 4-(2-(naphthalen-1-yl)quinoline-4-carboxamido)benzoate

  • Compound

    (R)-N-(Biphenyl-2-yl)-2-(4-(4-methoxyphenylsulfonyl)piperazin-1-yl)propanamide

  • Compound

    N-(3-(Benzyl(isopropyl)amino)propyl)-4-(2-chloro-6-fluorobenzyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide

  • Compound

    (R)-(4-Chlorophenyl)(4-(3-(4-(2-fluorophenyl)piperazin-1-yl)-2-hydroxypropoxy)phenyl)methanone

  • Compound

    1-((4-(4-Benzhydrylpiperazine-1-carbonyl)thiazol-2-yl)methyl)-3-(4-chlorophenyl)-1-isopropylurea

  • Compound

    (R)-N-(3-Cyano-6-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-(4-(2,3-dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazin-1-yl)acetamide

  • Compound

    (E)-N-(3-(4-Cinnamylpiperazin-1-yl)-3-oxopropyl)-2-methyl-N-((tetrahydrofuran-2-yl)methyl)furan-3-carboxamide

  • Compound

    1-(4-(3-(1,1-Difluoroethyl)benzyl)piperazin-1-yl)-3-(naphthalen-2-ylsulfonyl)propan-1-one

  • Compound

    1-((4-(4-Benzhydrylpiperazine-1-carbonyl)thiazol-2-yl)methyl)-3-(2-fluorophenyl)-1-isopropylurea

  • Compound

    (E)-(4-Cinnamylpiperazin-1-yl)(2-((5-methyl-4-p-tolyl-4H-1,2,4-triazol-3-ylthio)methyl)oxazol-4-yl)methanone

  • Compound

    2-(2-(4-(4-Fluorobenzoyl)piperazin-1-yl)ethylamino)-9,10-dimethoxy-6,7-dihydro-4H-pyrimido[6,1-a]isoquinolin-4-one

  • Compound

    (Z)-6-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazine-1-carbonyl)-2-(2-fluorobenzylidene)-4-methyl-2H-benzo[b][1,4]thiazin-3(4H)-one

  • Compound

    N-(9-Ethyl-9H-carbazol-3-yl)-2-(4-(4-methoxyphenylsulfonyl)piperazin-1-yl)acetamide

  • Compound

    2-(2-(4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylsulfonyl)piperazin-1-yl)acetamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide

  • Compound

    3-(Phenoxymethyl)-4-phenyl-1-((4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)methyl)-1H-1,2,4-triazole-5(4H)-thione

  • Compound

    (Z)-6-(4-(Benzo[d][1,3]dioxol-5-ylmethyl)piperazine-1-carbonyl)-2-(2-chlorobenzylidene)-4-methyl-2H-benzo[b][1,4]thiazin-3(4H)-one

  • Compound

    6-(4-(2-(Benzhydryloxy)ethyl)piperazine-1-carbonyl)-2-(4-chlorophenyl)-1,2-dihydropyridazin-3(4H)-one

  • Compound

    1-(3-((4-(4-Fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid

  • Compound

    2-(4-(2,5-Dichlorophenylsulfonyl)piperazin-1-yl)-N-(9-ethyl-9H-carbazol-3-yl)acetamide

  • Compound

    (S,E)-3-(4-(4-Cinnamylpiperazine-1-carbonyl)phenylamino)-2-ethylisoindolin-1-one

  • Compound

    (E)-1-((4-(4-Cinnamylpiperazine-1-carbonyl)thiazol-2-yl)methyl)-1-isopropyl-3-phenylurea

  • Compound

    (S)-1-((R)-2-Phenyl-1-(3-phenyl-1,2,4-oxadiazol-5-yl)ethylamino)-3-(4-(2-phenylpropan-2-yl)phenoxy)propan-2-ol

  • Compound

    Inositol 1,4,5-trisphosphate

  • Compound

    Cyclic ADP-ribose

  • Compound

    L-Tryptophan

  • Compound

    Lysotracker Red

  • Compound

    Nicotinic acid adenine dinucleotide phosphate acetoxymethyl ester

  • Compound

    D-Glucose

  • Compound

    Diazoxide

  • Compound

    Nicotinamide adenine dinucleotide reduced

  • Compound

    Nicotinamide adenine dinucleotide phosphate reduced

  • Compound

    4-Methoxybenzaldehyde

  • Compound

    3-(Chloromethyl)-4-methoxybenzaldehyde

  • Compound

    (S)-Methyl 1-(3-(chloromethyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate

  • Compound

    1-(2-Fluorophenyl)piperazine

  • Compound

    (S)-Methyl 1-(3-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate

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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.

Author information

Author notes

    • Edmund Naylor
    • , Abdelilah Arredouani
    •  & 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

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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.

Competing interests

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

Corresponding author

Correspondence to Grant C Churchill.

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    Supplementary Figures 1 and 2, Supplementary Scheme 1, Supplementary Table 1 and Supplementary Methods

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DOI

https://doi.org/10.1038/nchembio.150

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