Efficient Generation of Apo Sirt2 Crystals to Facilitate Investigation of Sirt2 Inhibitor Interactions

The selectivity pocket is a key binding site for selective inhibitors of the NAD+-dependent lysine deacylase Sirtuin 2 (Sirt2), a promising drug target due to its involvement in diseases like cancer and neurodegeneration. This pocket typically opens only upon binding of long fatty-acylated substrates or selective inhibitors. While crystal soaking could facilitate the structure-guided design of human Sirt2 (hSirt2) inhibitors, 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 to the active site of Sirt2. This necessitates resource-intensive co-crystallization to obtain Sirt2-inhibitor complexes. Here, we developed a method to rapidly produce high-quality Sirt2 apo crystals with an open selectivity pocket, suitable for high-throughput soaking experiments with small molecules. Screening the Maybridge Ro3 library using fluorescence polarization and thermal shift assays, we generated three novel Sirt2-fragment structures. These crystals demonstrated the ability to bind small molecules at the acyl-lysine channel entrance and the cofactor binding site, further validated by soaking with the peptide-based Sirt2 inhibitor KT9 and NAD+, respectively. These findings highlight the broad applicability of our unprecedented hSirt2 apo crystals, supporting structure-activity relationship studies and accelerating the design, development, and optimization of new Sirt2 inhibitor scaffolds.