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.