Role of Fluorination in the Histone Deacetylase 6 (HDAC6) Selectivity of Benzohydroxamate-Based Inhibitors

Abstract

Nonselective histone deacetylase (HDAC) inhibitors show dose-limiting side effects due to the inhibition of multiple, essential HDAC subtypes that can be limited or prevented by restricting their selectivity. We herein report the crystal structures of zebrafish HDAC6 catalytic domain 2 (zHDAC6-CD2) in complex with the selective HDAC6 inhibitors ITF3756 and ITF3985 and shed light on the role of fluorination in the selectivity of benzohydroxamate-based structures over class I isoforms. The reason for the enhancement in the selectivity of the benzohydroxamate-based compounds is the presence of specific interactions between the fluorinated linker and the key residues Gly582, Ser531, and His614 of zHDAC6, which are hindered in class I HDAC isoforms by the presence of an Aspartate that replaces Ser531. These results can be used in the design and development of novel, highly selective HDAC6 inhibitors.

Figure 1

Representative examples of selective HDAC6 inhibitors including the two internally developed HDAC6 inhibitors, ITF3756 and ITF3985. For all inhibitors, it is possible to distinguish the ZBG in red, the spacer in blue, the cap in green, and the ramification in black.

Figure 2

ACY-1083 (purple) and NR-160 (green) in the zHDAC6-CD2 binding site. The Zn ion interactions with zHDAC6 and the inhibitor ZBG are shown as thin gray lines. The picture shows the L1 pocket in terms of the Connolly surface. The light red surface indicates a relatively large crevice (pocket L1), involving Leu712, His463, Pro464, and Phe583; the light green surface indicates the L2 pocket, involving Asn530, Ser531, Phe642, and Phe643.

Figure 3

ITF3756 (A) and ITF3985 (B) in zHDAC6 CD2. The Zn ion is represented as a gray ball. HBs (green dashed lines), π–π interactions (magenta dashed lines), and a π-alkyl interaction (purple dashed line) are recognized.

Figure 4

Bavarostat (orange carbon atoms) superimposed with the crystal structure of compound 2-FF (magenta carbon atoms) in zCD2. The fluorinated ring of Bavarostat penetrates deeper into the catalytic tunnel, showing bidentate hydroxamate-Zn²⁺ coordination.

Figure 5

(A) Overlay of ITF3756 (1-H, green carbon atoms) and ITF3985 (2-FF, magenta carbon atoms) complexed with zCD2. The centroids of the phenyl substructures are not superimposable; the fluorinated aryl moiety lies 0.45 Å closer to the Zn ion. (B) Overlay of ITF3756 (green carbon atoms) and Bavarostat (orange carbon atoms) (PDB code 6DVO) complexed with zCD2. The center of mass displacement is larger (0.76 Å) with the monofluorinated inhibitor buried closer to the metal ion.