Abstract
Tissue nonspecific alkaline phosphatase (TNAP) is one of four human alkaline phosphatases (AP), a family of exocytic enzymes that catalyze hydrolysis of phospho-monoesters in bone, liver, kidney, and various other tissues. Overexpression of TNAP gives rise to excessive bone and soft tissue mineralization, including blood vessel calcification. Our prior screening campaigns have found several leads against this attractive therapeutic target using in vitro assay with a recombinant enzyme; these compounds were further optimized using medicinal chemistry approaches. To prioritize compounds for their use in animal models, we have designed and developed a biomarker assay for in situ detection of TNAP activity within human and mouse blood samples at physiological pH. This assay is suitable for screening compounds in 1,536-well plates using blood plasma from different mammalian species. The user may choose from two different substrates based on the need for greater assay simplicity or sensitivity.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Markello TC, Pak LK, St Hilaire C et al (2011) Vascular pathology of medial arterial calcifications in NT5E deficiency: implications for the role of adenosine in pseudoxanthoma elasticum. Mol Genet Metab 103:44–50
Addison WN, Azari F, Sorensen ES et al (2007) Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity. J Biol Chem 282:15872–15883
Sergienko EA, Millan JL (2010) High-throughput screening of tissue-nonspecific alkaline phosphatase for identification of effectors with diverse modes of action. Nat Protoc 5:1431–1439
Sergienko E, Su Y, Chan X et al (2009) Identification and characterization of novel tissue-nonspecific alkaline phosphatase inhibitors with diverse modes of action. J Biomol Screen 14:824–837
Chung TD, Sergienko E, Millan JL (2010) Assay format as a critical success factor for identification of novel inhibitor chemotypes of tissue-nonspecific alkaline phosphatase from high-throughput screening. Molecules 15:3010–3037
Dahl R, Sergienko EA, Su Y et al (2009) Discovery and validation of a series of aryl sulfonamides as selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP). J Med Chem 52:6919–6925
Sidique S, Ardecky R, Su Y et al (2009) Design and synthesis of pyrazole derivatives as potent and selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP). Bioorg Med Chem Lett 19:222–225
Coburn SP, Mahuren JD, Jain M et al (1998) Alkaline phosphatase (EC 3.1.3.1) in serum is inhibited by physiological concentrations of inorganic phosphate. J Clin Endocrinol Metab 83:3951–3957
Sergienko EA (2012) Basics of HTS assay design and optimization. In: Fu H (ed) Chemical genomics. Cambridge University Press, New York, pp 159–172
Ramaiah SK (2007) A toxicologist guide to the diagnostic interpretation of hepatic biochemical parameters. Food Chem Toxicol 45:1551–1557
Babson AL, Greeley SJ, Coleman CM et al (1966) Phenolphthalein monophosphate as a substrate for serum alkaline phosphatase. Clin Chem 12:482–490
Morgenstern S, Kessler G, Auerbach J et al (1965) An automated p-nitrophenylphosphate serum alkaline phosphatase procedure for the AutoAnalyzer. Clin Chem 11:876–888
Stinson RA (1993) Kinetic parameters for the cleaved substrate, and enzyme and substrate stability, vary with the phosphoacceptor in alkaline phosphatase catalysis. Clin Chem 39:2293–2297
Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73
Acknowledgments
This work is supported by NIH Roadmap grant # U54 HG005033 and Conrad Prebys Center for Chemical Genomics at Sanford-Burnham Medical Research Institute.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Sergienko, E.A., Sun, Q., Ma, CT. (2013). A Method for Direct Assessment of Tissue-Nonspecific Alkaline Phosphatase (TNAP) Inhibitors in Blood Samples. In: Millán, J. (eds) Phosphatase Modulators. Methods in Molecular Biology, vol 1053. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-562-0_6
Download citation
DOI: https://doi.org/10.1007/978-1-62703-562-0_6
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-561-3
Online ISBN: 978-1-62703-562-0
eBook Packages: Springer Protocols