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 (IC₅₀ = 2 nM). TO-317 exhibits 158-fold selectivity for HDAC6 over other HDAC isozymes by binding the catalytic Zn²⁺ and, uniquely, making a never seen before direct hydrogen bond with the Zn²⁺ 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.

Figure 1:

Currently known HDAC6 inhibitors. The structural features involve a hydroxamic acid which is required for Zn²⁺ binding, and a linker that connects the hydroxamic acid to a cap group. a) Currently known inhibitors achieved HDAC6 selectivity by replacing the benzene substituent on SAHA with a bulkier, usually hydrophobic cap group. b) Previous studies showed compound 1 is a nM inhibitor against HDAC6 exhibiting limited selectivity c) Current studies show TO-317 adopts a rotatable cap group with two aromatic substituents that occupy the HDAC6 surface facilitating specific residue interactions. *IC₅₀ values were determined using an activity-based electrophoretic mobility shift assay (EMSA) by Nanosyn Inc., USA.

Figure 2:

Overlaid Inhibition curves for TO-317 against a panel of 11 Zn²⁺-based HDAC homologs. TO-317 shows in vitro HDAC6 selectivity across all Zn²⁺-dependent HDAC homologs and becomes a viable candidate for further biological investigation of the HDAC6 enzyme.

Figure 3:

Docking conformation of TO-317 with drHDAC6 CD2 (PDB 5WGI) reveals occupancy of both L1 and L2 crevices which facilitates a plethora of interactions. Deck A shows the pocket view of these interactions, while deck B shows how TO-317 adopts a windmill conformation that allows both aromatic ring cap groups to engage with the L1 and L2 crevices of the HDAC6 outer surface.

Figure 4.

Stereo-view of a Polder omit map (contoured at 4.0σ) showing the binding of TO-317 in the active site of HDAC6 (PDB code: 7JOM). The catalytic Zn²⁺ ion is shown as a grey sphere, the Zn²⁺ bound water molecule is shown as a red sphere; metal coordination and hydrogen bond interactions are shown as solid and dashed black lines, respectively.

Figure 5:

Cellular potency of TO-317 and Citarinostat (positive control) in 12 cell lines (1 heathy cell, MRC-9 included). TO-317 shows anti-proliferative potency across different cancer cells with minimal activity in healthy cells. IC₅₀ values are indicated above each graph and reported in μM. IC₅₀ values with their 95% confidence intervals are reported in Table S3.

Figure 6:

TO-317 shows superior HDAC6 selectivity and potency in MV4–11 and MM.1S cells. (A) MV4–11 cells treated with increasing doses (0.25 μM - 1 μM) of Citarinostat and TO-317 show accumulation of Ac-α-tubulin for TO-317 at 0.25 μM but not for Citarinostat. (B) Similar dose-dependent responses were observed for both inhibitors in MM.1S cells.

Figure 7:

Quantification of Ac-α-tubulin and Ac-histone levels in immunofluorescence assay. DMSO was used as a negative control in each cohort, and Citarinostat was the positive control. Result indicates that TO-317 induces a clear dose-dependent increase in acetylated α-tubulin with minimal cellular accumulation of acetylated histones under the same dosing concentrations. Data reported above are an average of duplicate experiments.

Figure 8:

MV4–11 treated with TO-317 (Panel A) and Citarinostat (Panel B) leads to dose-dependent programmed cell death. Death by necrosis (Q1) is minimal and consistent at all doses. Dose-dependent cell death by TO-317 is consistent with a mechanistic approach of cell cycle arrest leading to apoptosis and corroborates the previously observed superior anti-proliferative activity of TO-317 over Citarinostat, the positive control. Data reported above are an average of duplicate experiments, and quantification of each cell population with the associated standard deviation is reported graphically in Fig. S8-A & Fig. S8-B.

Figure 9:

ROS generation in MV4–11 following incubation with 10 μM TO-317 for up to 4 h. TO-317 shows markedly increased levels of ROS after 2 h incubation which continues to increase up to the 4 h incubation time tested. Data reported is an average of duplicate studies.

Figure 10:

TO-317 is stable in vivo with a t_{1/2 (avg)} = 1.7 h. Male BALB/c mice were dosed TO-317 intraperitoneally (50 mg/mL solution 5% DMSO, 30% PEG400, 1% Tween80 & 64% saline) and peaks were detected and quantified from collected blood samples at different time concentrations using a triple-quad LC-MS/MS.