Detecting ZDHHC acyltransferase inhibition using acylation-coupled lipophilic induction of polarization (Acyl-cLIP)
Wenzhe Chen1, Jiaqi Zhao1, Zhenquan Sun2
1Department of Medicine, The University of Chicago, Chicago, IL, United States; Department of Chemistry, The University of Chicago, Chicago, IL, United States; Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, United States.
Abstract:
Acylation of protein substrates by ZDHHC (zinc finger Asp-His-His-Cys) acyltransferases is a key regulator of cell signaling. Potent and selective small molecule inhibitors for ZDHHCs have been long desired for the field but so far still do not exist. Robust activity assay methods are needed to discover selective ZDHHC inhibitors. Here we present two complementary assays for in vitro ZDHHC activity assay and inhibitor profiling. The Acylation-Coupled Lipophilic Induction of Polarization (Acyl-cLIP) assay is a homogeneous fluorescence polarization (FP) assay that uses a fluorophore-tagged peptide substrate; upon palmitoylation by ZDHHC enzymes, the substrate partitions into detergent micelles, increasing FP signal. This assay enables real-time kinetic measurements in 384-well plates suitable for high-throughput screening. As an orthogonal validation method, we use liquid chromatography-mass spectrometry (LC-MS) to directly detect palmitoylation through a +238 Da mass shift on model peptide substrates, allowing more reliable quantification and confirmation. Together, these methods offer a platform for identifying and validating ZDHHC inhibitors.
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