A story about difluoromethyl-1,3,4-oxadiazoles: Synthesis and biochemical evaluation of an enigmatic novel zinc binding group for the highly selective inhibition and degradation of histone deacetylase 6

Abstract

Histone deacetylase 6 (HDAC6) is a valuable pharmaceutical target in oncological and non-oncological diseases. Classical selective HDAC6 inhibitors utilize a hydroxamic acid as zinc binding group (ZBG), which is associated with mutagenic and genotoxic properties. Therefore, there is an urgent need for alternative ZBGs, such as the emerging 2-difluoromethyl-1,3,4-oxadiazole (DFMO) motif with an unprecedented HDAC6 selectivity profile. In this thesis, three projects are described, focusing on the development and elucidation of DFMOs as ZBG for selective HDAC6 inhibition and degradation. <br /> The first project covers the development of the first non-hydroxamate selective HDAC6 degraders. A set of six proteolysis targeting chimeras (PROTACs) was designed, synthesized, and characterized utilizing the DFMO warhead for HDAC6 recognition, bearing various polyethylenglycol and alkyl chain linkers, and containing E3 ligase ligands to recruit cereblon and von Hippel-Lindau. All degraders revealed highly selective HDAC6 inhibition and degradation. The dependence of degradation on ternary complex formation was confirmed, as well as degradation via the ubiquitin proteasome system. <br /> The second project discovers DFMOs as selective, mechanism-based, and essentially irreversible inhibitors of HDAC6. New HDAC6 inhibitors with DFMOs as ZBG were designed, synthesized, and evaluated for their inhibitory activity. To elucidate the binding mechanism, the hit compound was further investigated, including crystallographic experiments. The crystal structure revealed that the DFMO undergoes an enzyme catalyzed hydrolysis ring-opening reaction. The resulting anionic acylhydrazide acts as the active species and indicated tight-binding properties in jump dilution experiments. Kinetic binding studies confirmed that the reaction follows a two-step slow-binding mechanism. <br /> The third project summarizes in a viewpoint the future of selective HDAC6 modulation by DFMOs. The challenges and opportunities of DFMO inhibition and degradation were discussed, along with the history of the development of DFMOs as ZBGs for selective HDAC inhibition. In detail, binding mechanism studies and pharmacokinetic profiles were evaluated to highlight the advantages and disadvantages of this new class of selective HDAC6 inhibitors. <br /> In conclusion, this thesis provides new insights into the development of highly selective HDAC6 inhibitors and degraders utilizing DFMOs as a promising, alternative ZBG. For the first time, the enigmatic binding mechanism of DFMOs with unparalleled selective HDAC6 inhibition was disclosed. Finally, to highlight the potential of DFMOs as important drug candidates, the milestones in the development of DFMO compounds to selectively modulate HDAC6 were outlined.