Cloning, expression and purification of HDAC6 protein samples
DNAs encoding HDAC61109-1213 and HDAC61109-1215 were both subcloned into a modified pET28 vector encoding a thrombin cleavable (GenBank EF442785) N-terminal His6-tag (pET28-LIC) and HDAC61109-1215 was also subcloned into a modified pET28 vector encoding an N-terminal AviTag for in vivo biotinylation and a C-terminal His6-tag (p28BIOH-LIC). Subcloning was performed using ligation-independent InFusion™ cloning kit (ClonTech) and verified by DNA sequencing. All proteins were over-expressed in a BL21 (DE3) Codon Plus RIL E. coli strain (Agilent). Cultures were grown in M9 minimal media supplemented with 50 µM ZnSO4 and 10 µg/mL biotin for HDAC61109-1215 p28BIOH-LIC expression. Expression cultures were induced using 0.5 mM IPTG overnight at 15 °C. Proteins were purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (Qiagen) and the His6-tag was removed by thrombin protease for HDAC61109-1213 pET28-LIC. Uncleaved proteins and thrombin were removed by another pass with Ni-NTA resin. Proteins were further purified using gel filtration (Superdex 75, GE Healthcare). The final concentrations of purified proteins were 5-10 mg/mL as measured by UV absorbance at 280 nm.
DNA encoding HDAC61-1215 was cloned into a modified derivative pFastBac Dual vector (Invitrogen) encoding an N-terminal AviTag and a C-terminal His6 tag (pFBD-BirA). Cloning was completed using a ligation-independent InFusionTM cloning kit (ClonTech) and verified by DNA sequencing. HDAC61-1215 was over-expressed in sf9 insect cells. Cultures were grown in HyQ SFX Insect Serum Free Medium (Fisher Scientific) to a density of 4x106 cells/mL and infected with 10 mL of P3 viral stock media per 1 L of cell culture. Cell culture medium was collected after 4 days of incubation in a shaker. Protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) agarose resin (Qiagen). Following overnight dialysis, protein was further purified using anion exchange (HiTrap Q HP, GE Healthcare) and gel filtration (Superdex 75, GE Healthcare). The final concentration of purified HDAC61-1215 was 3.5 mg/mL as measured by UV absorbance at 280 nm.
Crystallisation and structure determination of HDAC6-ligand cocrystals
The apo crystal form of HDAC61109-1215 (PDB ID: 3C5K) was previously reported and can be obtained by mixing the protein solution at 3.5 mg/mL 1:1 with 3.5 M sodium formate, 0.1 M bis tris propane, 5 % (v/v) ethylene glycol, pH 7 using the vapor diffusion method at room temperature. These crystals can be used to seed for the soaking amenable crystal form for fragment screening. Diluting a 1 µL drop of these crystals 1:10,000 with mother liquor and vortexing the sample vigorously yields a seed mix. HDAC61109-1213 can then be crystallized in a high salt condition containing 2 M Na formate, 0.1 M Na acetate pH 4.6, 5 % ethylene glycol, again by the vapor diffusion method. 500 nL of protein are added to 400 nL mother liquor and then 100 nL seed mix per drop, typically plates are set up using a Mosquito (TTP LabTech). These crystals have a solvent exposed ubiquitin binding pocket, amenable to soaking. For all compounds, HDAC61109-1213 crystals were soaked by adding 5 % (v/v) of a 200 mM or 400 mM DMSO-solubilized stock of each compound to the drop for 2 hours prior to mounting and cryo-cooling.
X-ray diffraction data for HDAC61109-1213 crystals soaked with SGC-T094 or SGC-T164 were collected at 100K at Rigaku FR-E Superbright home source at a wavelength of 1.54178 Å. HDAC61109-1213 crystals soaked during LabXChem pipeline were collected at 100K at i04-1, Diamond Light Source (Harwell, U.K.) at a wavelength of 0.92819 Å, hit fragment datasets were identified using a DIMPLE-PANDDA pipeline. All datasets were processed with XDS and Aimless. Cycles of COOT, for model building and visualization, with REFMAC, for restrained refinement, were used to refine the models. Final models were validated with MOLPROBITY.
In vitro assays
Surface Plasmon Resonance (SPR): SPR studies were performed using a Biacore T200 (GE Health Sciences). Approximately 5000 response units (RU) of biotinylated HDAC61109-1215 were coupled onto one flow cell of a SA chip as per manufacturer’s protocol, while an empty flow cell was used for reference subtraction. 2-fold serial dilutions for all compounds were prepared in 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005 % (v/v) Tween-20 and 1 % (v/v) DMSO. KD determination experiments were performed using single-cycle kinetics with a contact time of 30 s and a flow rate of 30 µL/min at 20 °C. KD values were calculated using steady state affinity fitting and the Biacore T200 Evaluation software (GE Health Sciences).
Fluorescence Polarisation (FP) RLRGG Peptide Displacement: All experiments were performed in a total volume of 10 µL in 384-well black polypropylene PCR plates (Axygen). Fluorescence polarization (FP) was measured using a BioTek Synergy 4 (BioTek) after 10 min of incubation. The excitation and emission wavelengths were 485 nm and 528 nm, respectively. In each well, 9 µL 2 mM compound solutions in buffer containing 10 mM HEPES pH 7.4, 150 mM NaCl and 1 % (v/v) DMSO were diluted in the same buffer. 1 µL 30 µM HDAC61109-1215 or 10 µM HDAC61-1215 and 500 nM N-terminally FITC-labelled RLRGG were then added to each well. Following 1 in centrifugation at 250 g, the assay was incubated for 10 min before FP analysis.
Isothermal Titration Calorimetry (ITC): HDAC61109-1215 was diluted to 50 µM in 10 mM HEPES pH 7.4, 150 mM NaCl and 1 % (v/v) DMSO and all compounds were diluted to 1 mM in the same buffer. All ITC measurements were performed at 25 °C on a Nano ITC (TA Instruments). A total of 25 injections, each of 2 µL, were delivered into a 0.167 mL sample cell at a 180-second interval. The data were analyzed using Nano Analyze software and fitted to a one-site binding model.