A novel screening system to identify small-molecule inhibitors of S-acyltransferases

CONSUELO CORONEL; GIOLITO MARIA LUZ; IRIS ALEJANDRA GARCIA; GASTÓN SORIA; JAVIER VALDEZ TAUBAS

Salta

Congreso; 55 SAIB; 2019

SAIB

Protein S-acylation or palmitoylation, is a post-translational modification (PTM) that consist in the addition of long-chain fatty acids on cysteine residues through a thioester bond. The labile nature of this bond makes this PTM the only lipid modification that is reversible and therefore with regulatory capabilities. Palmitoylation has multiple roles in many cellular processes, including signal transduction, protein traffic and even gene expression. This modification is highly prevalent and more than 10% of the human proteome is thought to be palmitoylated. Additionally, there is growing evidence that palmitoylation is closely linked to human health. For example its misregulation has been associated to many types of cancers and disorders of the nervous system, and many viral and bacterial pathogens require palmitoylation by the host machinery to thrive, making the enzymes responsible for this modification attractive drug targets. Palmitoylation is catalyzed by a family of Palmitoyltransferases (PATs) or zDHHC proteins, which are polytopic membrane proteins characterized by the presence of a conserved DHHC-Cysteine Rich domain. There are 7 members of this family in yeast and 23 in humans. Despite the importance of palmitoylation, there no specific inhibitors of mammalian DHHC proteins are currently available. There is a great need to develop such inhibitors to aid in the study of this modification, and to test their possible therapeutic implications. The aim of this work is to develop an in vivo screening system to identify putative small-molecule inhibitors for PATs. We describe the creation of yeast strains that enable us to positively select the cells in which palmitoylation by an individual PAT is inhibited. This system is based on a reporter gene (HIS3) that responds to a transcription factor fused to a palmitoylation substrate. We show that the system works well for the endogenous yeast PAT Akr1 and it can be adapted for the isolation of inhibitors of heterologous PATs, both from mammalian and parasite origin, such as, human DHHC20, mouse DHHC21 and Giardia lamblia GL50803_6733. We are currently screening a 10.000 compound library (3D Biodiversity by ChemDiv) and have several candidate molecules for further validation.