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 Compounds that selectively inhibited palmitoylation of Nmyristoylated 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.

Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains

“…2-BP is a putative inhibitor of palmitoyl acyl transferases. 20 Treatment of MDA-MB-231 cells with 2-BP for 10 min resulted in the disappearance of the band visualized by conjugation with biotin (Fig. 1B, top).…”

“…We utilized 2-BP, which has been shown to be an inhibitor of palmitoyl acyl transferases. [20][21][22] Pretreatment of HEK293T cells transfected with WT-NTSR-1 with 10 μM 2-BP for 30 min significantly NTSR-1 were serum-starved for 24 h before 4 h ( 3 h) palmitate labeling. Cell lysates were subjected to immunoprecipitation using anti-ha antibody.…”

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

“…6). To determine whether palmitoylation has a role in activating endogenous ENaCs in mCCD cl1 cells, we treated polarized cultures of mCCD cl1 cells mounted in an Ussing chamber with either 2-bromopalmitate (2-BP), a general irreversible inhibitor of protein palmitoylation, or DMSO (vehicle control) (42)(43)(44). We observed a significant decrease in short circuit current (I sc ) within 5 min after apical addition of 2-BP that reached a new steady state I sc after 30 min (Fig.…”

Drug target identification at the crossroad of neuronal apoptosis and survival

“…Zdhhc8, in particular, influences mitochondria-regulated apoptosis when overexpressed [69]. Chemical inhibitors of palmitoylation, such compound V and 2BP [70], may be used to test the direct involvement of these proteins in neuronal apoptosis and survival.…”