title: Inhibition of formyl peptide-stimulated phospholipase D activation by Fal-002-2 via blockade of the Arf6, RhoA and protein kinase C signaling pathways in rat neutrophils.
abstract: Three recently developed selective phospholipase D (PLD) inhibitors N-(2-(4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)-2-naphthamide (VU0155056), (S)-N-(1-(4-(5-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)propan-2-yl)-2-naphthamide (VU0155069), and N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]decan-8-yl)ethyl)quinoline-3-carboxamide (VU0285655-1) inhibited O2 (•-) generation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel 2-phenyl-4-quinolone compound 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), which inhibited O2 (•-) generation, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC50 16.0 ± 5.0 μM). Fal-002-2 attenuated the interaction of PLD1 with ADP-ribosylation factor (Arf) 6, Ras homology (Rho) A and protein kinase C (PKC) isoforms (α, βI, and βII), and also inhibited the membrane recruitment of Arf6 and RhoA in fMLP-stimulated neutrophils, but not in GTPγS-stimulated cell-free system. The cellular levels of GTP-bound Arf6 and GTP-bound RhoA were reduced by Fal-002-2. Fal-002-2 also attenuated the membrane recruitment of Rho-associated protein kinase 1, phosphorylation of myosin light chain 2 at Thr18/Ser19 and PLD1 at Thr147, and the interaction of Arf6 with both arfaptin 1 and phosphatidylinositol 4-phosphate 5-kinase 1A. The association between RhoA and Vav, the interaction of Vav with both Lyn and Lck, the membrane recruitment of Vav, and the phosphorylation of Vav at Tyr174, but not Src family at Tyr416, were all attenuated by Fal-002-2 in fMLP-stimulated neutrophils. These results indicate that Fal-002-2 is not a direct PLD inhibitor, but the inhibition of fMLP-stimulated PLD activity by Fal-002-2, which partly accounts for its suppression of O2 (•-) generation, is attributable to the blockade of both Arf6 and RhoA activation and attenuation of the interaction of Arf6, RhoA and PKC isoforms with PLD1 in rat neutrophils.
authors.affiliation: Department of Medical Research, Taichung Veterans General Hospital, 160, Section 3, Chung Kang Road, Taichung, 407, Taiwan.
authors.forename: Ya-Ru
authors.initials: YR
authors.lastname: Tsai
authors.forename: Li-Jiau
authors.initials: LJ
authors.lastname: Huang
authors.forename: Hui-Yi
authors.initials: HY
authors.lastname: Lin
authors.forename: Yun-Jie
authors.initials: YJ
authors.lastname: Hung
authors.forename: Miau-Rong
authors.initials: MR
authors.lastname: Lee
authors.forename: Sheng-Chu
authors.initials: SC
authors.lastname: Kuo
authors.forename: Mei-Feng
authors.initials: MF
authors.lastname: Hsu
authors.forename: Jih-Pyang
authors.initials: JP
authors.lastname: Wang
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011804:Quinolines / Q000494:pharmacology; D015363:Quinolones / Q000008:administration & dosage / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects*; D013141:Spiro Compounds / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C582906:6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate; D000089982:ADP-Ribosylation Factor 6; D001562:Benzimidazoles; C585271:N-(1-(4-(5-chloro-2-oxo-2,3-dihydro-1H-benzo(d)imidazol-1-yl)piperidin-1-yl)propan-2-yl)-2-naphthamide; C585270:N-(2-(4-(2-oxo-2,3-dihydro-1H-benzo(d)imidazol-1-yl)piperidin-1-yl)ethyl)-2-naphthamide; C585272:N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro(4,5)decan-8-yl)ethyl)quinoline-3-carboxamide; D010880:Piperidines; D011804:Quinolines; D015363:Quinolones; D013141:Spiro Compounds; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D010739:Phospholipase D; C106801:phospholipase D1; D020727:ADP-Ribosylation Factors; C000715349:Arf6 protein, rat; D020742:rhoA GTP-Binding Protein