Control of the signaling of RAS proteins by modulating their palmitoylation

Small GTPases are key regulators of cell proliferation and oncogenesis. S-palmitoylation catalyzed by ZDHHC enzymes regulates many small GTPases, such as RAS family proteins, by modulating their subcellular localization. The development of chemical tools to modulate S-palmitoylation remains challenging due to the redundancy and poor druggability of ZDHHC enzymes. Here, we developed an approach to modulate RAS palmitoylation by fusing a depalmitoylase, acyl-protein thioesterase 1 (APT1), to the N-terminus of RAS proteins. S-palmitoylation, and thus the subcellular localization of RAS in the fusion proteins, can be reversibly controlled by an APT1 inhibitor, ML348. Using this approach, we developed a cell-based high-throughput assay to screen small-molecule regulators of NRAS palmitoylation and identified six compounds that inhibit the ZDHHC9-GCP16 complex, which catalyzes RAS palmitoylation, with IC₅₀ values ranging from 1.4 to 8.0 μM. Thus, our approach provides a useful tool for studying S-palmitoylation and screening regulators of this important post-translational modification.