Structure-Based Design of HDAC6 Inhibitors Targeting the L2 Pocket: Modulating Autophagy and Liquid−Liquid Phase Separation in Lung Cancer

Abstract We herein describe the structure-based design, synthesis and biological evaluation of a new class of histone deacetylase 6 (HDAC6) inhibitors guided by X-ray crystal structures of our previously identified compounds 13-15. X-ray co-crystal structures of second-generation inhibitors 19b (Subseries I) and 20b (Subseries II) within zHDAC6 CD2 confirmed that these compounds behave as highly potent HDAC6 inhibitors with the ability to engage the S531 residue in the L2 loop HDAC6 catalytic domain. Western-blot analysis confirmed the target engagement in cell-based settings, through the increase of acetylated tubulin levels, without significant variation in the histone acetylation status. Compound 20b extensively reduced the viability of A549 cells in both 2- and 3-dimensional cell cultures, through the activation of pro-autophagic pathways. In addition, HDAC6 inhibition impaired the nucleation of stress-granules, confirming the ability of these compounds to modulate liquid−liquid phase separation, an emerging HDAC6-driven hallmark in cancer.