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,Maurine E Linder

doi:10.1194/jlr.m800270-jlr200

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.

Highlights

Pharmacologic approaches to studying palmitoylation are limited by the lack of specific inhibitors
The pattern of specificity shown by the inhibitors in assays using cell membranes as a source of palmitoyltransferase activity suggests that some of the inhibitors would more potently inhibit DHHC proteins that modify farnesylated substrates, whereas others would be more effective inhibitors of protein acyltransferase (PAT) for N-myristoylated substrates
As advances are made in the palmitoylation field, it is becoming increasingly clear that DHHC proteins play an important role in native and disease states

Summary

Introduction

Pharmacologic approaches to studying palmitoylation are limited by the lack of specific inhibitors. Compounds that selectively inhibited palmitoylation of Nmyristoylated vs farnesylated peptides were identified in assays of palmitoyltransferase activity using cell membranes. We evaluated the ability of these inhibitors to reduce DHHC-mediated palmitoylation using purified enzymes and protein substrates. Human DHHC2 and yeast Pfa were assayed with their respective N-myristoylated substrates, Lck and Vac. 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. 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. Work in the authorslaboratories is supported by NIH grants GM51466 and NS057112 (M.E.L.), CA050211 and GM073977 (R.J.D.), and GM48099 (M.L.H)

Methods

Results

Conclusion