Switching the Switch: Ligand Induced Disulfide Formation in HDAC8

Abstract

Human histone deacetylase 8 is a well-recognized target for T-cell lymphoma and particularly childhood neuroblastoma. PD-404,182 was shown to be a selective covalent inhibitor of HDAC8 that forms mixed disulfides with several cysteine residues and is also able to transform thiol groups to thiocyanates. Moreover, HDAC8 was shown to be regulated by a redox switch based on the reversible formation of a disulfide bond between cysteines Cys₁₀₂ and Cys₁₅₃ . This study on the distinct effects of PD-404,182 on HDAC8 reveals that this compound induces the dose-dependent formation of intramolecular disulfide bridges. Therefore, the inhibition mechanism of HDAC8 by PD-404,182 involves both, covalent modification of thiols as well as ligand mediated disulfide formation. Moreover, this study provides a deep molecular insight into the regulation mechanism of HDAC8 involving several cysteines with graduated capability to form reversible disulfide bridges.

Keywords: HDAC8; covalent inhibitors; cysteine; redox switch; sulfenamides.

Figure 1

Mechanism of PD‐404,182 induced disulfide formation.

Figure 2

Structural overview of the investigated Cysteine pairs in HDAC8 (PDB‐ID: 1T64).

Figure 3

Structural alignment of HDAC1, 2, 3, 4, 6, 7 and 8. Cysteines are marked in black and disulfides in HDAC8 relate to black lines. Theoretical peptide fragments are indicated with grey bars beneath the alignment. Numbers on the left and right edge indicates the amino acid numbers corresponding to following PDB IDs: HDAC1 (4BKX), HDAC2 (5IWG), HDAC3 (4A69), HDAC4 (2VQJ), HDAC6 (5W5K), HDAC7 (3C0Z), HDAC8 (1T69).

Figure 4

Electrophoretic mobility shift assay (EMSA) showing disulfide bond induced loss of enzyme activity. A) EMSA for the HDAC8_wt, B) HDAC8_C102S/C153S, C) HDAC8_{C102S/C153S/C275S/C352S} enzyme.

Figure 5

HPLC‐MS/MS analysis of disulfide linked tryptic HDAC8 fragments. A) HPLC chromatogram of the untreated and PD‐404,182 treated HDAC8 samples. B) Mass spectra for the Cys₁₂₅‐S‐S‐Cys₁₃₁₋S‐CAM, C) Cys₁₂₅‐S‐S‐Cys₁₃₁+Cys₁₀₂‐S‐S‐Cys₁₅₃, D) Cys₂₄₄‐S‐S‐Cys₂₈₇, E) Cys₂₇₅‐S‐S‐Cys₃₅₂ peptide fragment.

Figure 6

Thermal shift assay of HDAC8 incubated with various concentrations of PD‐404,182. The black lines show the increase of SYPRO orange fluorescence upon thermal unfolding of HDAC8. The dotted lines represent the first derivatives of thermograms. Vertical dotted lines correspond to the melting point of the untreated control sample.