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

Salaun, Christine and Takizawa, Hiroya and Galindo, Alex and Munro, Kevin R. and McLellan, Jayde and Sugimoto, Isamu and Okino, Tomotaka and Tomkinson, Nicholas C.O. and Chamberlain, Luke H. (2022) Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation. Journal of Biological Chemistry, 298 (10). 102469. ISSN 1083-351X (https://doi.org/10.1016/j.jbc.2022.102469)

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Abstract

Protein S-acylation is a reversible post-translational modification that modulates the localization and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC (Asp-His-His-Cys) domain–containing (zDHHC) proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyze S-acylation in a two-step process involving "autoacylation" 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. Here, we developed and implemented a novel FRET-based high-throughput assay for the discovery of compounds that interfere with autoacylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. Our screen of >350,000 compounds identified two related tetrazole-containing compounds (TTZ-1 and TTZ-2) that inhibited both zDHHC2 autoacylation and substrate S-acylation in cell-free systems. These compounds were also active in human embryonic kidney 293T cells, where they inhibited the S-acylation of two substrates (SNAP25 and PSD95 [postsynaptic density protein 95]) mediated by different zDHHC enzymes, with some apparent isoform selectivity. Furthermore, we confirmed activity of the hit compounds through resynthesis, which provided sufficient quantities of material for further investigations. The assays developed provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating cellular activities of zDHHC enzymes in S-acylation.

ORCID iDs

Salaun, Christine ORCID logoORCID: https://orcid.org/0000-0003-3002-6810, Takizawa, Hiroya, Galindo, Alex, Munro, Kevin R., McLellan, Jayde, Sugimoto, Isamu, Okino, Tomotaka, Tomkinson, Nicholas C.O. ORCID logoORCID: https://orcid.org/0000-0002-5509-0133 and Chamberlain, Luke H. ORCID logoORCID: https://orcid.org/0000-0002-8701-4995;
CORE (COnnecting REpositories)