Methods for the kinetic analysis of time-dependent HDAC6 inhibitors

The class IIb histone deacetylase HDAC6 is responsible for the deacetylation of multiple cytoplasmic substrates, including α-tubulin and Miro-1, and has emerged as an important therapeutic target in neuropathies. In this context, several HDAC6 inhibitors effective in neuropathic models, exhibit slow-binding and slow-release behavior, thereby requiring kinetic approaches that extend beyond commonly used endpoint assays, which can lead to misinterpretation of inhibitor potency and mechanism of action. Notably, compounds with slow dissociation kinetics may offer improved therapeutic properties in neuropathic conditions, as sustained target engagement can translate into prolonged improvement of axonal transport and cytoskeletal dynamics. In this chapter, we outline a comprehensive methodology for the study of HDAC6 inhibition and the identification of slow-binding and slow-dissociating inhibitors.