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Molecules 2010, 15(5), 3010-3037; doi:10.3390/molecules15053010
Review

Assay Format as a Critical Success Factor for Identification of Novel Inhibitor Chemotypes of Tissue-Nonspecific Alkaline Phosphatase from High-Throughput Screening

1
, 1
 and 2,*
1 Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA 2 Sanford Children’s Health Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA
* Author to whom correspondence should be addressed.
Received: 20 January 2010 / Revised: 18 March 2010 / Accepted: 22 April 2010 / Published: 27 April 2010
(This article belongs to the Special Issue High-throughput Screening)
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Abstract

The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PPi), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.
Keywords: diethanolamine (DEA); absorption spectroscopy; luminescence; high throughput screening; CDP-Star®; Molecular Libraries; tissue-nonspecific alkaline phosphatase; alkaline phosphatase; chemical library; para-nitrophenylphosphate diethanolamine (DEA); absorption spectroscopy; luminescence; high throughput screening; CDP-Star®; Molecular Libraries; tissue-nonspecific alkaline phosphatase; alkaline phosphatase; chemical library; para-nitrophenylphosphate
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Chung, T.D.; Sergienko, E.; Millán, J.L. Assay Format as a Critical Success Factor for Identification of Novel Inhibitor Chemotypes of Tissue-Nonspecific Alkaline Phosphatase from High-Throughput Screening. Molecules 2010, 15, 3010-3037.

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