A Novel Zinc Binding Group for HDAC6 Inhibition

Abstract

Histone deacetylases (HDAC’s) are key regulatory enzymes in gene transcription and cellular motility through the deacetylation of lysine residues. These enzymes bear a distinct clinical significance, as the upregulation of HDACs has been associated with oncogenesis for many hematological malignancies and proliferation of other neurodegenerative or immune disorders. There are four different classes of HDACs, three of which require zinc for catalysis. Among the zinc dependent isozymes, HDAC6 is thought to be a particularly desirable therapeutic target, as its selective inhibition in malignant cells is accompanied by fewer associated toxicities in comparison to pan‐HDAC inhibition. Therefore, optimizing HDAC6 inhibitor selectivity has the potential to yield great clinical significance. This selectivity is often conferred by designing inhibitors with bulky capping groups and a hydrophobic linker region, which make favorable interactions in the hydrophobic region of the HDAC6 active site. Many inhibitors contain a hydroxamate zinc‐binding group, which can coordinate with bidentate or monodentate geometry. In efforts to optimize HDAC6 selectivity by exploring alternative zinc‐binding groups, a unique inhibitor containing an oxadiazole ring was discovered. Surprisingly, the crystal structure of its complex with HDAC6 reveals that the oxadiazole undergoes a ring opening reaction to yield an acylhydrazide that binds with an extended conformation in the active site.

Difluoromethyl-1,3,4-oxadiazoles are selective, mechanism-based, and essentially irreversible inhibitors of histone deacetylase 6

“…[47,48] In 2022, we reported on a DFMO derivative that underwent a ring opening reaction in the presence of HDAC6 leading to a acylhydrazide which was successfully co-crystallized in an extended conformation in the active site of HDAC6. [49] Shortly after our initial disclosure Steinkühler and co-workers observed a similar ring opening reaction followed by a hydrolysis reaction yielding a hydrazide derivative which was crystallized in complex with HDAC6. [50] Herein, we report the full experimental details of our initial conference abstract demonstrating that difluoromethyl-1,3,4-oxadiazoles act as selective, mechanism-based, and essentially irreversible inactivators capable of inhibiting HDAC6 via a two-step slow-binding mechanism.…”

Difluoromethyl-1,3,4-oxadiazoles Are Selective, Mechanism-Based, and Essentially Irreversible Inhibitors of Histone Deacetylase 6

“…However, the mechanism by which DFMOs inhibit or degrade HDAC6 remained enigmatic. In a conference abstract published in 2022, we disclosed that a DFMO derivative underwent an enzyme-catalyzed ring-opening reaction, resulting in an acylhydrazide that was cocrystallized in an extended conformation within the active site of HDAC6 . More recently, Barinka and co-workers conducted a comparative assessment of a hydroxamate-based HDAC6 inhibitor and its corresponding DFMO analogue.…”

“…Herein, we report the full experimental details of our 2022 conference abstract demonstrating that DFMOs act as selective, mechanism-based, and essentially irreversible inactivators capable of inhibiting HDAC6 via a two-step slow-binding mechanism. Our findings reveal that the zinc-bound water attacks the sp 2 carbon nearest to the difluoromethyl moiety of the DFMO group followed by a subsequent ring opening of the oxadiazole, thereby yielding a deprotonated difluoroacetylhydrazide as active species.…”

2-(Difluoromethyl)-1,3,4-oxadiazoles: The Future of Selective Histone Deacetylase 6 Modulation?

“…In a 2022 conference abstract, the Christianson group, in collaboration with us, disclosed that the DFMO derivative BK-1 (Figure C) underwent an enzyme-catalyzed ring-opening reaction . This process led to the formation of an acylhydrazide, which was subsequently co-crystallized in an extended conformation within the active site of HDAC6 .…”

“…In a 2022 conference abstract, the Christianson group, in collaboration with us, disclosed that the DFMO derivative BK-1 (Figure C) underwent an enzyme-catalyzed ring-opening reaction . This process led to the formation of an acylhydrazide, which was subsequently co-crystallized in an extended conformation within the active site of HDAC6 . In 2023, Steinkühler and colleagues elucidated the HDAC6 complex structure with a hydrazide inhibitor derived from the twofold hydrolysis of the DFMO inhibitor ITF5924 (Figure C) .…”