In this study, we utilised a difluoromethyl-1,3,4-oxadiazole zinc-binding group to develop the first non-hydroxamate selective HDAC6 degraders (PROTACs).

Histone deacetylases (HDACs) play a key role in the control of transcription, cell proliferation, and migration. FDA-approved histone deacetylase inhibitors (HDACi) demonstrate clinical efficacy in the treatment of different T-cell lymphomas and multiple myeloma. However, due to unselective inhibition, they display a wide range of adverse effects. One approach to avoiding off-target effects is the use of prodrugs enabling a controlled release of the inhibitor in the target tissue. Herein, we describe the synthesis and biological evaluation of HDACi prodrugs with photo-cleavable protecting groups masking the zinc-binding group of the established HDACi DDK137 (I) and VK1 (II). Initial decaging experiments confirmed that the photocaged HDACi pc-I could be deprotected to its parent inhibitor I. In HDAC inhibition assays, pc-I displayed only low inhibitory activity against HDAC1 and HDAC6. After irradiation with light, the inhibitory activity of pc-I strongly increased. Subsequent MTT viability assays, whole-cell HDAC inhibition assays, and immunoblot analysis confirmed the inactivity of pc-I at the cellular level. Upon irradiation, pc-I demonstrated pronounced HDAC inhibitory and antiproliferative activities which were comparable to the parent inhibitor I. Additionally, only phototreated pc-I was able to induce apoptosis in Annexin V/PI and caspase-Glo 3/7 assays, making pc-I a valuable tool for the development of light-activatable HDACi.

The development of selective histone deacetylase inhibitors (HDACi) and thus the identification of suitable chemical probes is still highly complex. Moreover, in the past, the results of oversimplified biochemical HDAC assays have often been overinterpreted. This has led to the extensive use of various supposedly isoform-selective HDACi as chemical probes to investigate the biological function of specific HDAC isoforms and classes. Considering more recent insights concerning the structure, binding kinetics, as well as substrate specificity of several HDAC isoforms, at least some of these studies should be reevaluated thoroughly. In this review, we present a selection of possible tool compounds for use in biological and pharmacological studies to investigate the biological function of specific HDAC isoforms or classes and comprehensively discuss their limitations based on the currently available data.