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 due to its involvement in diseases like cancer and neurodegeneration. While small molecule soaking could advance inhibitor development, the selectivity pocket is absent in current Sirt2 apo structures, and existing soaking systems like Sirt2-ADPribose (ADPR) suffer from disadvantageous crystal contacts that hinder ligand binding. We developed a method to rapidly generate high-quality Sirt2 apo crystals with an open selectivity pocket, suitable for high-throughput soaking experiments. The pocket formation is induced by seeding with a Sirtuin Rearranging ligand (SirReal) and is retained in the final apo structure without the presence of the ligand. Screening the Maybridge Ro3 library using fluorescence polarization and thermal shift assays yielded three novel Sirt2-fragment structures. These crystals can accommodate ligands at the acyl-lysine channel entrance and the cofactor binding site, as further confirmed with KT9 and NAD+, facilitating SAR studies and the optimization of inhibitors.