A Fluorescent Nucleosome Platform for High-Throughput Sirtuin Kinetic Analysis and Inhibitor Discovery

The class IV sirtuins SIRT6 and SIRT7 are epigenetic protein lysine deacylases identified as targets for cancer treatment, including pancreatic ductal adenocarcinoma (PDAC). However, small molecules to study their regulatory roles or modulate their function in disease remain scarce. A major reason is that class IV sirtuins require nucleosomal substrates to be active, and current peptide-based assays therefore fail as tools for activity profiling and inhibitor development. Here, we develop a translatable kinetic assay platform by engineering a fluorescence-based activity sensor directly into semi-synthetic nucleosomes. This enables activity profiling and screening of modulators of epigenetic eraser enzymes. For both SIRT6 and SIRT7, we measure enzyme kinetics in real time, yielding an assay with nanomolar Michaelis-Menten substrate constants and Z’ values over 0.8. For SIRT7, we provide a mechanistic model of nucleosomal substrate selectivity and activity regulation, and for SIRT6, we use the platform to assay hits selected from an in-cell DNA-encoded library screening campaign. As a result, we identify vpc03898 as a potent, selective, and cell-permeable competitive inhibitor of SIRT6 with activity against PDAC cell migration at single-digit µM concentration. Together, this work establishes a translatable strategy for kinetic screening of nucleosome-targeting eraser enzymes toward cancer therapeutic development.