title: Fluorescence probe for lysophospholipase C/NPP6 activity and a potent NPP6 inhibitor.
abstract: 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-mPC(3)C, TG-mPC(5)C, TG-mPENE, TG-mPEA, TG-mPhos, TG-mPA, TG-mPMe, and TG-mPPr. Among the seven NPPs, only NPP6 hydrolyzed TG-mPC, TG-mPC(3)C, 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 80,000 compounds. Through structure-activity relationship (SAR) analysis, we identified a potent and selective NPP6 inhibitor with an IC(50) value of 0.21 μM. Our NPP6-selective fluorescence probe, TG-mPC, and the inhibitor are expected to be useful to elucidate the biological function of NPP6.
authors: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan.
authors: Mitsuyasu
authors: M
authors: Kawaguchi
authors: Takayoshi
authors: T
authors: Okabe
authors: Shinichi
authors: S
authors: Okudaira
authors: Kenjiro
authors: K
authors: Hanaoka
authors: Yuuta
authors: Y
authors: Fujikawa
authors: Takuya
authors: T
authors: Terai
authors: Toru
authors: T
authors: Komatsu
authors: Hirotatsu
authors: H
authors: Kojima
authors: Junken
authors: J
authors: Aoki
authors: Tetsuo
authors: T
authors: Nagano
mesh_terms: D000818:Animals; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008245:Lysophospholipase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D015394:Molecular Structure; D041681:NIH 3T3 Cells; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D008245:Lysophospholipase; D010727:Phosphoric Diester Hydrolases; C502079:ENPP6 protein, human