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High-throughput screening of tissue-nonspecific alkaline phosphatase for identification of effectors with diverse modes of action

Nature Protocols volume 5, pages 1431–1439 (2010)Cite this article

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Abstract

Here we describe a protocol for the identification of effectors of tissue-nonspecific alkaline phosphatase (TNAP). It is based on a highly sensitive method for detecting TNAP activity. After dephosphorylation by TNAP, a dioxetane-based substrate undergoes a series of chemical transformations resulting in light production. Light intensity serves as a quantitative measure of the velocity of the TNAP-catalyzed reaction in the steady state. This protocol includes guidelines for optimizing the assay and for high-throughput screening in multiwell plates. The assay is sensitive to the influence of diverse effectors of TNAP as long as the assay optimization steps are repeated for each new batch of the enzyme; full optimization is accomplished in under 2 d. Depending on the available equipment, 10,000–100,000 compounds can be screened in an 8-h period. This protocol provides a method of screening TNAP that is 1,000-fold more sensitive and 10-fold faster than a conventional colorimetric assay with p-nitrophenyl phosphate.

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Figure 1: Catalytic mechanism of alkaline phosphatase reaction11.
Figure 2: Structures of two commonly used alkaline phosphatase substrates.
Figure 3: Luminescence-based assay for TNAP.
Figure 4: Optimization of CDP-Star concentration.
Figure 5: Optimization of DEA concentration.
Figure 6: Optimization of TNAP concentration in the luminescence assay.
Figure 7: Inhibition of TNAP activity by levamisole.
Figure 8: Z′-factor study of the optimized TNAP luminescence assay.

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Acknowledgements

This work was supported by NIH Roadmap Initiative Grant U54HG003916 (E.A.S.) and NIH Grant RC1 HL101899 (J.L.M.).

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Authors and Affiliations

  1. Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, La Jolla, California, USA

    Eduard A Sergienko

  2. Sanford Children's Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, California, USA

    José Luis Millán

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  1. Eduard A Sergienko
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Contributions

J.L.M. identified and validated TNAP as a potential therapeutic target. E.A.S. designed and optimized the assay and performed the screening. E.A.S. wrote the paper with guidance and discussion from J.L.M.

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Correspondence to Eduard A Sergienko.

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The authors declare no competing financial interests.

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Sergienko, E., Millán, J. High-throughput screening of tissue-nonspecific alkaline phosphatase for identification of effectors with diverse modes of action. Nat Protoc 5, 1431–1439 (2010). https://doi.org/10.1038/nprot.2010.86

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