Abstract
Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. BMS-229724 (4-[4-[2-[2-[bis(4-chlorophenyl)methoxy]ethyl-sulfonyl]ethoxy]phenyl]-1,1,1-trifluoro-2-butanone) was found to be a selective inhibitor of cPLA2(IC50 = 2.8 μM) in that it did not inhibit secreted phospholipase A2 in vitro, nor phospholipase C and phospholipase D in cells. The compound was active in inhibiting arachidonate and eicosanoid production in U937 cells, neutrophils, platelets, monocytes, and mast cells. With a synthetic covesicle substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 1 · 10−5 mol% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.35 mol%. The unit of concentration in the interface is mole fraction (or mol%), which is related to the surface concentration of substrate, rather than bulk concentration that has units of molarity. Thus, BMS-229724 represents a novel inhibitor of cPLA2, which partitions into the phospholipid bilayer and competes with phospholipid substrate for the active site. This potent inhibition of the enzyme translated into anti-inflammatory activity when applied topically (5%, w/v) to a phorbol ester-induced chronic inflammation model in mouse ears, inhibiting edema and neutrophil infiltration, as well as prostaglandin and leukotriene levels in the skin. In hairless guinea pigs, BMS-229724 was active orally (10 mg/kg) in a UVB-induced skin erythema model in hairless guinea pigs.
Footnotes
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1 This equation is valid, since the active site dissociation constant for DMPM has been shown to be more than 330 times greater than the value for PAPC (Burke et al., 1997d).
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2 KM*appand KI*app are related to the intrinsic dissociation constants (KM* andKI*) by the equations:KM*app =KM* (1 + 1/KL*) and KI*app =KI* (1 + 1/KL*), where KL* is the active site dissociation constant for DMPM at the interface (Burke et al., 1997d).
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3 When determining the equilibrium dissociation constants from the active site, the mole amount of substrate ([14C]PAPC in this case) and the mole amount of DMPM were held constant, while varying the mole amount of inhibitor (Burke et al., 1995b, 1997d). This has the effect of actually decreasing the mole fraction of both substrate and DMPM as the inhibitor concentration is increased. Thus,XSO in eq. 1 equals the mole fraction of substrate phospholipid without inhibitor. The mole fraction of substrate in the presence of the inhibitor is correspondingly less.
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4 The partition coefficient,C, is defined as the concentration (molarity) of inhibitor in the phospholipid bilayer divided by the concentration in the aqueous phase.
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↵5 In eq. 2, M is defined as:
- Abbreviations:
- PLA2
- phospholipase A2
- cPLA2
- cytosolic PLA2
- sPLA2
- secreted PLA2
- LPS
- lipopolysaccharide
- DMPM
- 1,2-dimyristoyl-sn-glycero-3-phosphomethanol
- PAPC
- 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine
- [14C]PAPC
- 1-palmitoyl-2-arachidonoyl-[arachidonoyl-1-14C]-sn-glycero-3-phosphocholine
- fMLP
- N-formyl-methionyl-leucyl-phenylalanine
- PLC
- phospholipase C
- PLD
- phospholipase D
- IL
- interleukin
- TNFα
- tumor necrosis factor-α
- LTB4
- leukotriene B4
- PGE2
- prostaglandin E2
- PAF
- platelet-activating factor
- TPA
- 12-O-tetradecanoylphorbol-13-acetate
- BisTris
- 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
- AACOCF3
- arachidonyl trifluoromethyl ketone
- NF-κB
- nuclear factor-κB
- AP-1
- activating protein-1
- AUC
- area under the curve
- i.p.t.
- intraportal
- Vdss
- steady-state volume of distribution
- Received February 2, 2001.
- Accepted March 22, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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