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

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 the S-acylation of 2 substrates (SNAP25 and PSD95) 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 zDHHC enzymes in S-acylation

Discovery of Novel Seven-Membered Prostacyclin Analogues as Potent and Selective Prostaglandin FP and EP3 Dual Agonists

A novel series of prostaglandin analogues with a seven-membered ring scaffold was designed, synthesized, and evaluated for the functional activation of prostaglandin receptors to identify potent and subtype-selective FP and EP3 dual agonists. Starting from the prostacyclin derivative <b>5b</b>, a nonselective agonist for prostaglandin receptors, replacement of the core structure with an octahydro-2<i>H</i>-cyclopenta­[<i>b</i>]­oxepine scaffold led to the discovery of the potent and selective FP and EP3 dual agonist <b>11b</b> as a lead compound for the development of an antiglaucoma agent