Abstract
The signaling enzyme phospholipase D (PLD) and the lipid second messenger it generates, phosphatidic acid (PA), are implicated in many cell biological processes, including Ras activation, cell spreading, stress fiber formation, chemotaxis, and membrane vesicle trafficking. PLD production of PA is inhibited by the primary alcohol 1-butanol, which has thus been widely employed to identify PLD/PA-driven processes. However, 1-butanol does not always effectively reduce PA accumulation, and its use may result in PLD-independent deleterious effects. Consequently, identification of potent specific small-molecule PLD inhibitors would be an important advance for the field. We examine one such here, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), which was identified recently in an in vitro chemical screen for PLD2 inhibitors, and show that it rapidly blocks in vivo PA production with subnanomolar potency. We were surprised to find that several biological processes blocked by 1-butanol are not affected by FIPI, suggesting the need for re-evaluation of proposed roles for PLD. However, FIPI does inhibit PLD regulation of F-actin cytoskeleton reorganization, cell spreading, and chemotaxis, indicating potential utility for it as a therapeutic for autoimmunity and cancer metastasis.
Footnotes
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This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grants GM071520, GM071475, GM50388, T32-GM008444]; the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL50040]; the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant F31-DK082280]; the National Institutes of Health National Center for Research Resources [Grants S10-RR024598, P20-RR021954]; the Turner Foundation; an American Heart Association Postdoctoral Fellowship; and the Carol M. Baldwin Breast Cancer Research Fund.
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ABBREVIATIONS: PLD, phospholipase D; PC, phosphatidylcholine; PA, phosphatidic acid; Ptd-But, phosphatidyl butanol; PIP2, phosphatidyl inositol 4,5-bisphosphate; FIPI, 5-fluoro-2-indolyl des-chlorohalopemide; HPLC, high-performance liquid chromatography; CHO, Chinese hamster ovary; FBS, fetal bovine serum; Dox, doxycycline; GFP, green fluorescent protein; DMSO, dimethyl sulfoxide; PAGE, polyacrylamide gel electrophoresis; ERK, extracellular signal-regulated kinase; DMSO, dimethyl sulfoxide; KRBH, Krebs-Ringer bicarbonate HEPES buffer; PMA, phorbol 12-myristate 13-acetate; RNAi, RNA interference.
- Received November 3, 2008.
- Accepted December 8, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), a Phospholipase D Pharmacological Inhibitor That Alters Cell Spreading and Inhibits Chemotaxis
