CONICET | Buscador de Institutos y Recursos Humanos

Protein S-acylation or palmitoylation is a post-translational modification (PTM) that consists of the addition of long-chainfatty acids on cysteine residues through a thioester bond. The labile nature of this bond makes this PTM reversible and,therefore, capable of exerting regulatory functions. Palmitoylation has multiple roles in many cellular processes, includingsignal transduction, protein traffic, and even gene expression. This modification is highly prevalent and more than 10% of thehuman proteome is thought to be palmitoylated. Palmitoylation is catalyzed by a family of palmitoyltransferases (PATs) orzDHHC proteins, which are polytopic membrane proteins characterized by the presence of a conserved DHHC-Cysteine RichDomain. There is growing evidence that palmitoylation is closely linked to human health. For instance, inappropriate activationof the epidermal growth factor receptor (EGFR) contributes to a variety of human malignancies, recently it has been shownthat silencing of the palmitoyltransferase DHHC20 creates a dependence on EGFR signaling for cancer cell survival, as aconsequence it enhances the vulnerability of the cells to an existing first line treatment for EGFR-driven cancers. Despite theimportance of palmitoylation, no specific inhibitors for DHHC proteins are currently available. There is a great need to developsuch inhibitors to aid in the study of this modification and to test their possible therapeutic implications. Here we show thedevelopment of a yeast-based in vivo high-throughput screening method for the identification of small molecules withinhibitory activity for different PATs. This system is based on a reporter gene (HIS3) that responds to a transcription factorfused to a palmitoylation substrate. When palmitoylation is inhibited, the cells are able to grow in media lacking histidine. Itis therefore a positive selection method, which avoids highly toxic molecules. We first screened 3200 compounds from theChemDiv 3D Biodiversity library to find inhibitors of the endogenous yeast PAT Akr1 and selected a candidate compoundwhich we are currently characterizing. We next adapted the screening method so it can be used to isolate inhibitors of thehuman PAT DHHC20. Since the crystal structure of DHHC20 is available, we carried out an in silico screening usingVINARDO scoring function, to select compounds with the best predicted binding energy, to the DHHC20 active site. Weanalyzed 10000 compounds from the 3D Biodiversity library and selected the top 100 scoring molecules. These selectedcompounds were then tested in the in vivo system, and two compounds allowed growth of yeast cells, suggesting that theymight act as DHHC20 inhibitors

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