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.

Fig. 1.

Structure and names of inhibitor compounds used in this study.

Fig. 2.

Purification of human DHHC2 and catalytically inactive DHHS2. DHHC2 was expressed in Sf9 cells and purified sequentially by nickel chelate and FLAG affinity chromatography. Catalytically inactive DHHS2 was purified by nickel affinity chromatography; the fifth elution is shown. Purified DHHC2 and DHHS2 were detected by Sypro staining (left) and immunoblotting with anti-Express antibody (center). DHHC2, but not DHHS2, autoacylates (right). DHHC2, DHHS2, or heat inactivated DHHC2 (500 fmol each) were incubated in 0.8 μM [³H]palmCoA at pH 6.4 for 10 min. The reactions were stopped with SDS sample buffer and processed for fluorography. The film was exposed for 8 days.

Fig. 3.

DHHC2 palmitoylates myrLck_NT at cysteines 3 and 5. Purified DHHC2, DHHS2 (200 fmol), or enzyme buffer was mixed with purified myrLck_NT (25 pmol) on ice. Reaction buffer was added to give a final concentration of 0.8 μM [³H]palmCoA in 50 μl and incubated at 25°C for 7 min. The reaction was stopped with SDS sample buffer and divided between two SDS-PAGE gels, which were stained and destained. A: The region containing myrLck_NT was excised from one gel, solubilized with Soluene, and the amount of [³H]palmitate present determined by scintillation counting. B: The second gel was processed for fluorography and exposed to film at −80°C for 90 h. MyrLck_NT was detected by immunoblotting reactions run without radioactive palmCoA.

Fig. 4.

Inhibitor profiles for DHHC proteins. Inhibitor profiles for each enzyme-substrate pair were carried out as described in the text. A: Partially purified yeast Pfa3 (5 μl) was preincubated with inhibitor for 8 min at 30°C. MyrVac8 and [³H]palmCoA were mixed, warmed to 30°C, and added to the reaction at final concentrations of 0.5 μM and 0.8 μM, respectively. The reaction proceeded for 10 min at 30°C before stopping with SDS sample buffer. B: Purified human DHHC2 (100 fmol) was preincubated 8 min at 25°C with inhibitors. MyrLck_NT and [³H]palmCoA were added to give final concentrations of 0.5 μM and 0.8 μM, respectively, and the reaction proceeded for 7 min at 25°C before stopping. C: Purified yeast Erf2/Erf4 (100 fmol) was preincubated 8 min at 25°C with inhibitors. [³H]palmCoA and the farnesylated hypervariable region of Ras2 fused to glutathione S-transferase (GST) were added to give final concentrations of 0.9 μM and 1.0 μM, respectively, and the reaction proceeded for 10 min at 25°C before stopping. D: Purified human DHHC9/GCP16 (250 fmol) was preincubated 10 min on ice with inhibitors. [³H]palmCoA and farnesylated H-Ras fused to GST were added to give final concentrations of 0.9 μM and 1.0 μM, respectively, and the reaction proceeded for 7 min at 25°C before stopping. Values represent the means ± SEM determined from three independent experiments.

Fig. 5.

Inhibitor effect on enzyme autoacylation. DHHC2 (250 fmol) was preincubated with inhibitor and assayed as described in Materials and Methods. The film was exposed for 8 days.

Fig. 6.

Reversibility and time-dependent inhibition. Experiments were performed as described in Materials and Methods. A: Reversibility of 2-bromopalmitate (2BP) and Compound V (CV) was tested by incubating DHHC2 (100 fmol) with the first concentration indicated for 30 min at 25°C. This mix was diluted 40-fold with both substrates to the postdilution concentration indicated, incubated at 25°C, and aliquots removed at the indicated time points. B: Time-dependent inhibition was evaluated by preincubating enzyme with inhibitors at the indicated concentrations or dimethylsulfoxide (DMSO) for 30 min at 25°C. Both substrates without or with inhibitor were added to start the reaction, which was incubated at 25°C for 7 min before stopping. Values represent the means ± SEM determined from three independent experiments.