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Papers In Press, published online ahead of print February 1, 2009
J. Lipid Res., doi:10.1194/jlr.M800270-JLR200

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Journal of Lipid Research, Vol. 50, 233-242, February 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro^*,

Benjamin C. Jennings^*, Marissa J. Nadolski^*, Yiping Ling, Meredith Beckham Baker, Marietta L. Harrison, Robert J. Deschenes and Maurine E. Linder^1,*

^* Department of Cell Biology and Physiology, Washington University in Saint Louis, School of Medicine, 660 South Euclid Ave, Box 8228, St. Louis, MO 63110
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, Hansen Life Science Building, 201 South University Street, West Lafayette, IN 47907

^* B.C.J. and M.J.N. were supported by American Heart Association pre-doctoral fellowships (Midwest Affiliate). Work in the authors' laboratories is supported by NIH grants GM51466 and NS057112 (M.E.L.), CA050211 and GM073977 (R.J.D.), and GM48099 (M.L.H).

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of a figure.

Published, JLR Papers in Press, September 30, 2008.

¹ To whom correspondence should be addressed. e-mail: mlinder{at}wustl.edu

Pharmacologic approaches to studying palmitoylation are limited by the lack of specific inhibitors. Recently, screens have revealed five chemical classes of small molecules that inhibit cellular processes associated with palmitoylation (Ducker, C. E., L. K. Griffel, R. A. Smith, S. N. Keller, Y. Zhuang, Z. Xia, J. D. Diller, and C. D. Smith. 2006. Discovery and characterization of inhibitors of human palmitoyl acyltransferases. Mol. Cancer Ther. 5: 1647–1659). Compounds that selectively inhibited palmitoylation of N-myristoylated vs. farnesylated peptides were identified in assays of palmitoyltransferase activity using cell membranes. Palmitoylation is catalyzed by a family of enzymes that share a conserved DHHC (Asp-His-His-Cys) cysteine-rich domain. In this study, we evaluated the ability of these inhibitors to reduce DHHC-mediated palmitoylation using purified enzymes and protein substrates. Human DHHC2 and yeast Pfa3 were assayed with their respective N-myristoylated substrates, Lck and Vac8. Human DHHC9/GCP16 and yeast Erf2/Erf4 were tested using farnesylated Ras proteins. Surprisingly, all four enzymes showed a similar profile of inhibition. Only one of the novel compounds, 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one [Compound V (CV)], and 2-bromopalmitate (2BP) inhibited the palmitoyltransferase activity of all DHHC proteins tested. Hence, the reported potency and selectivity of these compounds were not recapitulated with purified enzymes and their cognate lipidated substrates. Further characterization revealed both compounds blocked DHHC enzyme autoacylation and displayed slow, time-dependent inhibition but differed with respect to reversibility. Inhibition of palmitoyltransferase activity by CV was reversible, whereas 2BP inhibition was irreversible.

Supplementary key words DHHC, Asp-His-His-Cys • lipidation • fatty acylation • S-acylation • enzyme • inhibitor

Abbreviations: 2BP, 2-bromopalmitate; β-ME, β-mercaptoethanol; CBP, calmodulin binding peptide; CV, Compound V, 2-(2-Hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one; DDM, n-dodecyl-β-D-maltoside detergent; DHHC, Asp-His-His-Cys; DMSO, dimethylsulfoxide; DTT, 1,4-dithiothreitol; GST, glutathione S-transferase; htt, huntingtin; MBOAT, membrane-bound O-acyltransferase; myrLck_NT, N-myristoylated lymphocyte specific kinase, N-terminal residues 1-226; [³H]palmCoA, [³H]^9,10-palmitoyl coenzyme A; PAT, protein acyltransferase; PMSF, phenylmethylsulphonyl fluoride

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