Efficient crystallization of Apo Sirt2 for small-molecule soaking and structural analysis of ligand interactions

The selectivity pocket is a key binding site for inhibitors of the NAD+-dependent lysine deacylase Sirtuin 2 (Sirt2), a promising drug target in diseases like cancer. While small-molecule soaking can advance inhibitor development, the selectivity pocket is absent in available Sirt2 apo structures, and existing soaking systems like Sirt2–ADPribose (ADPR) suffer from unfavorable crystal packing that hinders ligand binding. We developed a method to rapidly generate high-quality Sirt2 apo crystals with an open selectivity pocket, suitable for high-throughput soaking. The induced-fit pocket forms upon seeding with a Sirtuin Rearranging ligand (SirReal) and is retained in the ligand-free apo structure. Screening the Maybridge Ro3-fragment library using a fluorescence polarization assay yielded three novel Sirt2-fragment-inhibitor structures. Additionally, our Sirt2 apo crystals can accommodate ligands at the acyl-lysine channel entrance and the cofactor binding site, as confirmed by binding of the peptide inhibitor KT9 and NAD+, facilitating SAR studies and inhibitor optimization.