Development of Acyl Protein Thioesterase 1 (APT1) Inhibitor Palmostatin B That Revert Unregulated H/N‐Ras Signaling

This chapter describes the combination of bioinformatics, organic synthesis, in vitro inhibition studies, and live-cell imaging (microscopy) to elucidate the function of acyl protein thioesterase 1 (APT1) in regulation of protein palmitoylation. APT1 critically influences the localization and function of several palmitoylated peripheral membrane proteins of the rat sarcoma (Ras) and Rous sarcoma oncogene cellular homolog (Src) family, which themselves have pivotal roles in cancer signaling. The chapter provides a case study in which protein structure similarity clustering (PSSC) was applied to find small-molecule inhibitors of the enzyme APT1. The inhibitors were applied in reverse chemical genetics investigations of Ras localization and signaling in cell-based studies. In order to determine the efficacy of palmostatin B to inhibit APT1 in cells, Fluorescence lifetime imaging microscopy (FLIM) was performed with TAMRA-labeled (tetramethyl-6-carboxyrhodamine) derivative of palmostatin B in cells expressing APT1-GFP.