Development of an Acrylamide-Based Inhibitor of Protein S-Acylation

Abstract

Protein S-acylation is a dynamic lipid post-translational modification that can modulate the localization and activity of target proteins. In humans, the installation of the lipid onto target proteins is catalyzed by a family of 23 Asp-His-His-Cys domain-containing protein acyltransferases (DHHC-PATs). DHHCs are increasingly recognized as critical players in cellular signaling events and in human disease. However, progress elucidating the functions and mechanisms of DHHC "writers" has been hampered by a lack of chemical tools to perturb their activity in live cells. Herein, we report the synthesis and characterization of cyano-myracrylamide (CMA), a broad-spectrum DHHC family inhibitor with similar potency to 2-bromopalmitate (2BP), the most commonly used DHHC inhibitor in the field. Possessing an acrylamide warhead instead of 2BP's α-halo fatty acid, CMA inhibits DHHC family proteins in cellulo while demonstrating decreased toxicity and avoiding inhibition of the S-acylation eraser enzymes, two of the major weaknesses of 2BP. Our studies show that CMA engages with DHHC family proteins in cells, inhibits protein S-acylation, and disrupts DHHC-regulated cellular events. CMA represents an improved chemical scaffold for untangling the complexities of DHHC-mediated cell signaling by protein S-acylation.

Figure 1.

In vitro discovery of CMA as an inhibitor of human zDHHC20. (A) Structure and IC₅₀ against human zDHHC20 of key molecules used in this work. (B) Fluorescence polarization (FP) screening of a panel of acrylamide-based molecules against zDHHC20 with activity normalized to DMSO. Data are presented as the mean ± standard deviation (n = 3).

Figure 2.

Characterization of CMA. (A) Incubation of purified APT1 or APT2 (50 nM) with either 2BP or CMA, followed by measurement of APT activity using the fluorogenic probe DPP-5. Data are presented as the mean ± standard deviation (n = 3). (B) Viability of HEK293T, MDA-MB-231, and 3T3-L1 cells treated with varied concentrations of either 2BP or CMA (24 h), as measured by the MTS assay. Data are presented as the mean ± standard deviation (n ≥ 3).

Figure 3.

Validation of CMA in cellulo. (A) HEK293T cells pretreated with DMSO, 2BP, or CMA (0, 5, 10, and 20 μM, 3 h) before treatment with 17-octadecynoic acid (17-ODYA) (6 h) to metabolically label palmitoylated proteins. Isolated proteomes were then subject to click chemistry to conjugate TAMRA-azide to proteins modified by 17-ODYA, followed by protein separation via SDS-PAGE. In-gel fluorescence revealed a proteome-wide decrease in protein palmitoylation with CMA treatment. (B) Acyl-biotin exchange (ABE) of HEK293T cells treated with 2BP or CMA (20 μM, 6 h). Global S-acylation visualized using silver staining with CNX as a loading control (n ≥ 2). (C) Dose-response change in the S-acylation of exogenous eGFP-tagged GobX or HA-tagged Myd88 and endogenous Ras in HEK293T cells upon CMA treatment (6 h) as measured by ABE, carried out in serum-free media. CNX serves as an assay and loading control (n ≥ 2). (D) Acyl-biotin exchange (ABE) of HEK293T cells expressing eGFP-tagged GobX and treated with 2BP or CMA (0, 20, 50 μM) carried out in serum-free or serum-full media.

Figure 4.

Engagement of CMA with DHHC family proteins. (A) In-gel fluorescence analysis of myc-tagged DHHC proteins overexpressed in HEK293T cells treated with CMA or DMSO (2 h), followed by labeling with 8 (1 μM, 2 h). Cell lysates subject to click chemistry to conjugate TAMRA-azide to proteins modified by 8, followed by protein separation via SDS-PAGE. Expression levels visualized via antimyc tag Western blot (n ≥ 2). (B) Western blot analysis of endogenous DHHC proteins in HEK293T cells treated with CMA or DMSO (3 h), followed by labeling with 8 (20 μM, 3 h). Cell lysates were subject to click chemistry to conjugate biotin-azide to proteins modified by 8, followed by streptavidin enrichment, protein separation via SDS-PAGE, and Western blotting for indicated targets ( n ≥ 2).

Figure 5.

Confirmation of CMA functional activity in cellulo. (A) Analysis of EGFR S-acylation via ABE upon CMA treatment (20 μM, 3 h) in MDA-MB-231 cells with CNX as an assay and loading control (n ≥ 2). (B) EGF (100 ng/mL, 15 min)-induced phosphorylation of AKT with and without CMA treatment (20 μM, 3 h) (n ≥ 2). (C) Analysis of CD36 S-acylation via ABE upon CMA treatment (20 μM, 6 h) in 3T3-L1 cells with CNX as an assay and loading control (n ≥ 2). (D) Representative images of fluorescence imaging of 3T3-L1 preadipocytes starved (12 h) and treated with DMSO or CMA (20 μM, 3 h), followed by 10 μM BSA-bound oleate and BODIPY493/503 or BODIPY-palmitate (2 μM, 3 h) (n = 5). Scale bar = 20 μM.