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. File list (2)download file view on ChemRxiv SA8_V28 changes accepted.pdf (16.08 MiB) download file view on ChemRxiv SI_Revision4.pdf (37.14 MiB)

A High-Throughput Fluorescent Turn-On Assay for Inhibitors of DHHC Family Proteins

“…Our laboratory recently developed an acrylamide-based DHHC inhibitor, cyanomyracrylamide (CMA), which has decreased cytotoxicity and an altered reactivity profile, compared to 2BP. However, similar to 2BP, CMA is a lipid-based molecule with limited selectivity that targets a broad spectrum of DHHC family proteins, making it unsuitable for probing the biology of individual DHHCs …”

“…However, similar to 2BP, CMA is a lipid-based molecule with limited selectivity that targets a broad spectrum of DHHC family proteins, making it unsuitable for probing the biology of individual DHHCs. 16 …”

“… 19 Further, the acylation-coupled lipophilic induction of polarization (acyl-cLIP) assay has been successfully adapted for zDHHC3, zDHHC7, and zDHHC20. 16 , 20 In this assay, a fluorophore-labeled peptide is palmitoylated by a DHHC enzyme and inserted into detergent micelles, resulting in a change in fluorescence polarization (FP). However, the dynamic range of this assay is limited, and the peptide substrate requirements for each DHHC isoform are not fully known.…”

“…While the coupled enzyme assay, which detects the release of CoA during the palmitoyl transfer process, is fluorescence-based and therefore suitable for HTS, the indirect readout renders it prone to false positives . Further, the acylation-coupled lipophilic induction of polarization (acyl-cLIP) assay has been successfully adapted for zDHHC3, zDHHC7, and zDHHC20. , In this assay, a fluorophore-labeled peptide is palmitoylated by a DHHC enzyme and inserted into detergent micelles, resulting in a change in fluorescence polarization (FP). However, the dynamic range of this assay is limited, and the peptide substrate requirements for each DHHC isoform are not fully known.…”

Protein Palmitoylation Modification During Viral Infection and Detection Methods of Palmitoylated Proteins

“…Although 2bromopalmitate had been recognized as an inhibitor of protein palmitoylation, it has widely been recognized to exert off-target effects by inhibiting other enzymes involved in lipid metabolism and is also cytotoxic to cells (Pedro et al, 2013). A new inhibitor of DHHC, cyanomyracrylamide (CMA), was reported recently, which has a broad-spectrum inhibitory effect on DHHC family members like 2-bromopalmitate but with less toxicity and off-target effects (Azizi et al, 2021). v. Analysis of the structures of enzymes and viral proteins involved in the palmitoylation/depalmitoylation cycle would be helpful for a better understanding of the subtle ways of host-virus interactions and designing new inhibitors or drugs.…”