Mechanistic and Structural Insights on Difluoromethyl-1,3,4-oxadiazole Inhibitors of HDAC6

Abstract

Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) kinetic experiments, comprehensive enzymatic characterizations, and X-ray crystallography, we dissect the intricate details of the DFMO-HDAC6 interaction dynamics. More specifically, we find that the chemical structure of a DMFO and the binding mode of its difluoroacetylhydrazide derivative are crucial in determining the predominant hydrolysis mechanism. Our findings provide additional insights into two different mechanisms of DFMO hydrolysis, thus contributing to a better understanding of the HDAC6 inhibition by oxadiazoles in disease modulation and therapeutic intervention.

Keywords: DFMO hydrolysis; LC-MS; NMR; X-ray crystallography; difluoroacetylhydrazide (DFAcH); difluoromethyl-1,3,4-oxadiazole (DFMO); enzyme kinetics; histone deacetylase 6 (HDAC6); non hydroxamic inhibitors.

Figure 1

Diagram illustrating the potential reaction pathways of DFMO hydrolysis by HDAC6. The figure depicts the binding of DFMO to the HDAC6 active site, the first hydrolysis reaction, and the formation of the tight complex between the enzyme and DFAcH. Two alternative mechanisms for DFAcH hydrolysis to hydrazide are presented: (mechanism 1) DFAcH release, entry of a catalytic water molecule from the solvent to restore the tetrahedral zinc coordination sphere and intermediate rebinding for second hydrolysis reaction; (mechanism 2) entry of the second catalytic water molecule without intermediate dissociation and direct hydrolysis to hydrazide. Legend: difluoromethyl-1,3,4-oxadiazole (DFMO, purple), difluoroacetylhydrazide (DFAcH, light purple), hydrazide (Hyd, yellow), the zinc ion (Zn, grey), and water (H₂O, light blue). Created with BioRender.com.

Figure 2

Structures of DFMO-selective HDAC6 inhibitors. (A) ITF5924, ITF6715, and ITF6712 are compounds 1, 2, and 3 from our previous publication [22]. (B) The DFMO-bearing compound ITF7209 and its acyl-hydrazide and hydrazide derivatives (ITF7738 and ITF7739), which are described for the first time in this work. The hydrazide group, the final metabolite of DFMO, is depicted in blue, while the intermediate difluoroacylhydrazide is in red.

Figure 3

Hydrolysis of ITF5924 by zHDAC6-CD2 as detected by NMR. Evolution of DFMO (black), DFAcH (red), and hydrazide (blue) concentrations and the DFAcH-to-hydrazide molar ratio (purple open circles) over time (0 to 60 h). The kinetic experiment was conducted using 5 μM enzyme and 2 mM DFMO in deuterated assay buffer (25 mM d-Tris-DCl, 0.5 mM d-Tris(2-carboxyethyl)phosphine, pH 8.0). The experiment was carried out in singlicate (N = 1).

Figure 4

Hydrolysis of ITF5924 and ITF7209 by zHDAC6-CD2 as detected by LC-MS. The time course of 5 μM ITF5924 (A) or 5 μM ITF7209 (C) consumption during incubation at 25 °C with zHDAC6-CD2 (5 μM). Experiments like those shown in panel A and C were carried out in the presence of 100 μM ITF3756, which was added after 1 min of ITF5924-enzyme (B) or ITF7209-enzyme (D) pre-incubation. All the experiments were carried out in triplicate. The lines simply join the average values of the experimental points. The incomplete recovery could be attributed to the relatively high enzyme concentration, possibly resulting in coprecipitation phenomena upon the addition of acetonitrile.

Figure 5

Structures of zHDAC6-CD2/DFAcH complexes. Comparison between the binding conformer of difluoroacetylhydrazide analogues of ITF7209 (panel (A), pdb code 9EU0) and compound 6 (panel (B), pdb code 8GD4 [26]).