β-Lactams SB 212047 and SB 216754 Are Irreversible, Time-Dependent Inhibitors of Coenzyme A-Independent Transacylase
- James D. Winkler1,
- Chiu-Mei Sung1,
- Marie Chabot-Flecher1,
- Don E. Griswold1,
- Lisa A. Marshall1,
- Floyd H. Chilton3,
- William Bondinell and
- Ruth J. Mayer1
- Departments of 1Immunopharmacology (J.D.W., C.-M.S., M.C.-F., D.E.G., L.A.M., R.J.M.) and 2Medicinal Chemistry (W.B.), SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406, and3Section on Pulmonary and Critical Care Medicine and Department of Biochemistry (F.H.C.), Bowman Gray School of Medicine, Winston-Salem, North Carolina 27103
Abstract
The enzyme coenzyme A-independent transacylase (CoA-IT) has been demonstrated to be the key mediator of arachidonate remodeling, a process that moves arachidonate into 1-ether-containing phospholipids. Blockade of CoA-IT by reversible inhibitors has been shown to block the release of arachidonate in stimulated neutrophils and inhibit the production of eicosanoids and platelet-activating factor. We describe novel inhibitors of CoA-IT activity that contain a β-lactam nucleus. β-Lactams were investigated as potential mechanism-based inhibitors of CoA-IT on the basis of the expected formation of an acyl-enzyme intermediate complex. Two β-lactams, SB 212047 and SB 216754, were shown to be specific, time-dependent inhibitors of CoA-IT activity (IC50 = 6 and 20 μm, respectively, with a 10-min pretreatment time). Extensive washing and dilution could not remove the inhibition, suggesting it was irreversible. In stimulated human monocytes, SB 216754 decreased the production of eicosanoids in a time-dependent manner. In an in vivo model of phorbol ester-induced ear inflammation, SB 216754 was able to inhibit indices of both edema and cell infiltration. Taken together, the results support two hypotheses: 1) CoA-IT activity is important for the production of inflammatory lipid mediators in stimulated cells andin vivo and 2) the mechanism by which CoA-IT acts to transfer arachidonate is through an acyl-enzyme intermediate.
Footnotes
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Send reprint requests to: Dr. James D. Winkler, Department of Immunopharmacology, UW-2532, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406. E-mail:james_d_winkler{at}sbphrd.com
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This work was supported in part by National Institute of Health Grants AI24985 and AI26771.
- Abbreviations:
- CoA-IT
- coenzyme A-independent transacylase
- AA
- arachidonic acid
- BSA
- bovine serum albumin
- GPC
- sn-glycero-3-phosphocholine
- 5LO
- 5-lipoxygenase
- PLA2
- phospholipase A2
- PBS
- phosphate-buffered saline
- PAF
- platelet-activating factor
- TLC
- thin layer chromatography
- HBSS
- Hanks’ balanced salt solution
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- LT
- leukotriene
- PG
- prostaglandin
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- Received June 11, 1997.
- Accepted October 31, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
