title: 2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro.
abstract: 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.
authors.affiliation: Department of Cell Biology and Physiology, Washington University in Saint Louis, School of Medicine, 660 South Euclid Ave, Box 8228, St. Louis, MO 63110, USA.
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Jennings
authors.forename: Marissa J
authors.initials: MJ
authors.lastname: Nadolski
authors.forename: Yiping
authors.initials: Y
authors.lastname: Ling
authors.forename: Meredith Beckham
authors.initials: MB
authors.lastname: Baker
authors.forename: Marietta L
authors.initials: ML
authors.lastname: Harrison
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Deschenes
authors.forename: Maurine E
authors.initials: ME
authors.lastname: Linder
mesh_terms: D000217:Acyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D054878:Lipoylation; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000494:pharmacology*; D010168:Palmitates / Q000494:pharmacology*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
chemical_list: C539980:2-(2-hydroxy-5-nitrobenzylidene)benzo(b)thiophen-3-one; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009596:Nitrophenols; D010168:Palmitates; D029701:Saccharomyces cerevisiae Proteins; D013876:Thiophenes; D025521:Tumor Suppressor Proteins; C022776:2-bromopalmitate; D000217:Acyltransferases; C532036:ZDHHC2 protein, human; C403284:ERF2 protein, S cerevisiae; C092131:SHR5 protein, S cerevisiae; C504825:ZDHHC9 protein, human; C050480:glycylpeptide N-tetradecanoyltransferase