Fluorescence Probe for Lysophospholipase C/NPP6 Activity and a Potent NPP6 Inhibitor

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• 作者：Mitsuyasu Kawaguchi ; Takayoshi Okabe ; Shinichi Okudaira ; Kenjiro Hanaoka ; Yuuta Fujikawa ; Takuya Terai ; Toru Komatsu ; Hirotatsu Kojima ; Junken Aoki ; Tetsuo Nagano
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：August 10, 2011
• 年：2011
• 卷：133
• 期：31
• 页码：12021-12030
• 全文大小：1056K
• 年卷期：v.133,no.31(August 10, 2011)
• ISSN：1520-5126

文摘

Nucleotide pyrophosphatases/phosphodiesterases (NPPs) are ubiquitous membrane-associated or secreted ectoenzymes that have a role in regulating extracellular nucleotide and phospholipid metabolism. Among the members of the NPP family, NPP1 and -3 act on nucleotides such as ATP, while NPP2, -6, and -7 act on phospholipids such as lysophosphatidylcholine and sphingomyelin. NPP6, a recently characterized NPP family member, is a choline-specific glycerophosphodiester phosphodiesterase, but its functions remain to be analyzed, partly due to the lack of highly sensitive activity assay systems and practical inhibitors. Here we report synthesis of novel NPP6 fluorescence probes, TG-mPC and its analogues TG-mPC₃C, TG-mPC₅C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC₃C, and TG-mPENE. TG-mPC was hydrolyzed in the cell lysate from NPP6-transfected cells, but not control cells, showing that it is suitable for use in cell-based NPP6 assays. We also examined the usefulness of TG-mPC as a fluorescence imaging probe. We further applied TG-mPC to carry out high-throughput NPP6 inhibitor screening and found several NPP6-selective inhibitors in a library of about 80鈥?00 compounds. Through structure鈥揳ctivity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC₅₀ value of 0.21 渭M. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.