Unique Molecular Interaction with the Histone Deacetylase 6 Catalytic Tunnel: Crystallographic and Biological Characterization of a Model Chemotype

Abstract

Histone deacetylase 6 (HDAC6) is involved in multiple regulatory processes, ranging from cellular stress to intracellular transport. Inhibition of aberrant HDAC6 activity in several cancers and neurological diseases has been shown to be efficacious in both preclinical and clinical studies. While selective HDAC6 targeting has been pursued as an alternative to pan-HDAC drugs, identifying truly selective molecular templates has not been trivial. Herein, we report a structure−activity relationship study yielding TO-317, which potently binds HDAC6 catalytic domain 2 (K i = 0.7 nM) and inhibits the enzyme function (IC 50 = 2 nM). TO-317 exhibits 158-fold selectivity for HDAC6 over other HDAC isozymes by binding the catalytic Zn 2+ and, uniquely, making a never seen before direct hydrogen bond with the Zn 2+ coordinating residue, His614. This novel structural motif targeting the second-sphere His614 interaction, observed in a 1.84 Å resolution crystal structure with drHDAC6 from zebrafish, can provide new pharmacophores for identifying enthalpically driven, high-affinity, HDAC6-selective inhibitors.

Structure-based prediction of HDAC6 substrates validated by enzymatic assay reveals determinants of promiscuity and detects new potential substrates

“…To assess if performance was affected by the specific crystal structure selected we repeated the analysis using different structures. The DD2 domain of HDAC6 was solved several times, bound to different ligands: (1) a cyclic peptide (PDB ID: 6WSJ 30 ) (2) a tripeptide substrate attached to coumarin (PDB ID: 5EFN 4 ), and (3) an inhibitor (PDB ID: 7JOM 33 ). Best performance was achieved with the cyclic peptide (6WSJ), while the structure with the inhibitory small molecule performed worst (Table 2 B and Supplementary Fig.…”

Development of HDAC Inhibitors Exhibiting Therapeutic Potential in T-Cell Prolymphocytic Leukemia

“…Coupling to pentafluorobenzenesulfonyl chloride (S4h) (78%) and following the aforementioned deprotection yielded 3-(((N- 1a) was screened against HDAC3, 6, 8, 11 (representative of groups I, II, and IV) to determine in vitro activity inhibition profiles (Figure 1b). 48,49 Concurrently, cellular activity was analyzed in a model cancer cell line (MV4-11) and healthy fibroblasts (MRC-9) to correlate biochemical inhibition with cellular potency and therapeutic window, and other HDACi (SAHA/vorinostat, ricolinostat, and citarinostat), as well as parent compound 1, were included for parallel comparison.…”

Discovery of HDAC6-Selective Inhibitor NN-390 with in Vitro Efficacy in Group 3 Medulloblastoma

“…Horseradish peroxidase (HRP)-conjugated goat antimouse IgG secondary antibody (7076, Cell Signaling) or HRP-linked anti-rabbit IgG secondary antibody (7074, Cell Signaling) were applied to the membrane (1:5000 dilution), and bands were visualized using clarity western ECL substrate luminal/enhancer solution and peroxide solution 1:1 ratio for HRP-conjugated secondary antibody (BioRad) and analyzed using Image Lab software (BioRad). 55,58 Maximum Tolerated Dose Studies. Acute toxicity of the relevant compound was studied using 4−12-week-old male CD-1 mice (Charles River).…”

“…Expression and purification experiments were performed as previously described. 55,58,83 The DNA sequence corresponding to the catalytic domain 2 (CD2) of zebrafish HDAC6 (NCBI Accession Number: XP_009302026. After ∼18 h, the cells were harvested by centrifugation, and the pellets were frozen at −80 °C until protein purification.…”