Identifying Autopalmitoylation Inhibitors Through Scaffold Ranking

Abstract

Protein palmitoylation is a reversible posttranslational lipidation that plays an important role in cellular signaling, protein localization, stability, and aggregation. The dysregulation of palmitoylation has been implicated in a multitude of diseases including cancer, neurological disorders, and infectious diseases. A family of zinc‐finger “zDHHC” enzymes characterized by a conserved Asp‐His‐His‐Cys (DHHC) tetrapeptide sequence mediates palmitoylation. Having a unique inhibitor specific to the zDHHC enzyme(s) could lead to novel disease therapeutics. Despite advances in our understanding of the genetics and enzymology the current best inhibitor of palmitoylation, 2‐bromopalmitic acid, lacks specificity and affinity for zDHHC enzymes. Our lab has developed an assay amenable to high throughput screening, which measures the rate of the first step of the palmitoylation reaction, autopalmitoylation. During autopalmitoylation there is a release of CoASH, which is then coupled to an alpha‐ketoglutarate dehydrogenase reaction to produce fluorescent NADH. The production of NADH occurs at zero‐order kinetics and directly correlates to the reduction of CoA, and thus to the rate of autopalmitoylation. The introduction of an inhibitor of autopalmitoylation to this reaction would cause a reduction in the rate of NADH production. This coupled assay was used to screen for inhibitors of palmitoylation from a unique chemical scaffold library and resulted in the identification of a number of compounds. These results were supported through the use of orthogonal and bio orthogonal autopalmitoylation assays to further elucidate the type of inhibition.