Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation

Abstract

Protein S-acylation is a reversible post-translational modification that modulates the localisation and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC domain-containing proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyse S-acylation in a two-step process involving "auto-acylation" of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. In this study, we developed and implemented a novel FRET-based high throughput assay for the discovery of compounds that interfere with auto-acylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. A screen of >350,000 compounds identified two related tetrazole containing compounds (TTZ-1 and -2) that inhibited both zDHHC2 auto-acylation and substrate S-acylation in cell-free systems. Furthermore, these compounds were also active in HEK293T cells, where they inhibited substrate S-acylation mediated by different zDHHC enzymes, with some apparent isoform selectivity. Resynthesis of the hit compounds confirmed their activity, providing sufficient quantities of material for further investigations. The assays developed herein provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating the cellular activities of S-acylation and zDHHC enzymes.