5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), a Phospholipase D Pharmacological Inhibitor That Alters Cell Spreading and Inhibits Chemotaxis

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.

A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity

“…secretion can by facilitated by PLD1 while this effect is opposed by PLD2 [22]. In the present study, Ca 2+ influx from the extracellular compartment was unaffected by FIPI.…”

“…PLD is a signaling enzyme that generates the second messenger PA implicated in many cellular processes like adhesion, integrin activation [28], cell spreading [29], chemotaxis [22] and Ras activation [30]. PA production by PLD is abrogated by primary alcohol derivates like ethanol or 1-butanol.…”

“…FIPI was reported to inhibit both PLD1 and PLD2 in a dose-dependent manner, with 50% loss of activity observed at approximately 25 nM [22]. Thus, FIPI is a potent, concentrationdependent PLD2 inhibitor that inhibits PLD1 equally well under in vitro assay conditions [22;34].…”

“…Recently the novel pharmacological PLD-inhibitor FIPI has been developed and characterized [22]. Most intriguingly, it was found that cellular responses applying 1-butanol or FIPI essentially differed, strongly suggesting that the proposed role(s) of PLD need(s) to be re-evaluated.…”

Regulation of Phospholipase D Activity and Phosphatidic Acid Production after Purinergic (P2Y6) Receptor Stimulation

“…PLD1 and PLD2 are differentially localized, with PLD1 being mostly found in intercellular membranes and PLD2 found constitutively at the plasma membrane (10). Our laboratory (16 -19) as well as others (20,57) have published several reports implicating small molecule PLD inhibitors as possible anti-cancer agents. It has been shown that PLD inhibitor treatment leads to decreased cancer cell invasion (16), cell spreading, chemotaxis (20), and possibly could be a tool to decrease cancer metastasis (58).…”