Small-Molecule Proteomimetic Inhibitors of the HIF-1α–p300 Protein–Protein Interaction

The therapeutically relevant hypoxia inducible factor HIF-1α–p300 protein–protein interaction can be orthosterically inhibited with α-helix mimetics based on an oligoamide scaffold that recapitulates essential features of the C-terminal helix of the HIF-1α C-TAD (C-terminal transactivation domain). Preliminary SAR studies demonstrated the important role of side-chain size and hydrophobicity/hydrophilicity in determining potency. These small molecules represent the first biophysically characterised HIF-1α–p300 PPI inhibitors and the first examples of small-molecule aromatic oligoamide helix mimetics to be shown to have a selective binding profile. Although the compounds were less potent than HIF-1α, the result is still remarkable in that the mimetic reproduces only three residues from the 42-residue HIF-1α C-TAD from which it is derived.


Standard Procedure A -Ester Hydrolysis
An aqueous sodium hydroxide solution (2 M, 1 mL per 100 mg of ester) was added to a solution of the ester in methanol (~5 mL per 100 mg of ester) and stirred at room temperature until the starting material had been consumed, as observed by TLC. The reaction mixture was concentrated by half in vacuo then adjusted to pH 3 by the addition of 1M HCl (aq.); the resulting precipitate was isolated by filtration, dried in vacuo and used without further purification in subsequent steps.

Standard Procedure B -Tin Mediated Nitro reduction
Tin (II) chloride dihydrate (5 equivalents) was added in one portion to a solution of the nitro compound in ethyl acetate (5 mL per 100 mg) and the reaction stirred at 50°C under a calcium chloride drying tube for 24 hours. The reaction was then allowed to cool to room temperature and poured into 2 M sodium hydroxide solution (5 ml per 100 mg of starting material). The organic layer was separated, washed with 2 M sodium hydroxide solution (2 × 5 mL per 100 mg of starting material) and brine (5 mL per 100 mg of starting material), dried over magnesium sulphate and concentrated in vacuo. The residue was purified by flash column chromatography to give the desired compound.

Standard Procedure C -Nitro reduction by hydrogenation
Palladium on charcoal (10 %) was added against a flow of nitrogen to a solution of the nitro compound in methanol (10 mL per 100 mg) under a nitrogen atmosphere, the atmosphere was then replaced with hydrogen and the reaction stirred vigorously until complete by TLC (typically 2 hours). NO  The hydrogen atmosphere was vented and the reaction mixture filtered through a pad of Celite with methanol, concentrated in vacuo and purified by flash column chromatography.

Standard Procedure D -Coupling
Dichlorotriphenylphosphorane (4.5 equivalents) was added to a solution of nitro-acid compound (1.2 equivalents) in chloroform (5 mL per 100 mg of amine) and the reaction heated to reflux with stirring under nitrogen. After 2 hours at reflux, the amine ester compound (1 equivalent) was added as solution in chloroform (1 mL) and the reaction was heated to reflux for a further 24 hours. The reaction mixture was then concentrated in vacuo and partitioned between ethyl acetate (5 mL per 100 mg of amine) and H 2 O (5 mL per 100 mg of amine). The organic layer was separated and washed with saturated aqueous sodium bicarbonate solution (5 mL per 100 mg of amine), dried over magnesium sulphate and concentrated in vacuo. The resulting residue was purified by flash column chromatography to give the desired compound.

LIBRARY SYNTHESIS
The compound library was prepared in parallel using the methods and building blocks described above, checking at pertinent times during the synthesis by crude NMR and LC-MS, to afford the below compounds. Compounds were either pure following final precipitation or purified by preparative HPLC. Where the amount of material produced allowed 13 C-NMR spectra were collected otherwise 1 H-NMR and high resolution mass spectrometry were used to characterise the compound library.         Compound 13 was synthesised previously [

methyl]aniline
Prepared using a modified literature procedure, [4] aniline (1 mL, 10.6 mmol) was added to a solution

P300 (330 -420)
pGEX-p300 was transformed in to BL21 (DE3) pLysS Gold cells and incubated at 37°C with aeration until an OD600 0.6-0.8 was reached. Protein expression was induced by addition of 1mM sterile IPTG to the culture and the culture was then incubated at 18°C with aeration overnight in the presence of 50 µM Zinc. Cells were harvested by centrifugation at 3500 × g at 4°C for 20 minutes. Cell pellets were resuspended in Lysis buffer (20 mM TRIS pH 7.9, 500 mM NaCl, 0.1% (v/v) triton X-100). 5 U DNase I and 2 mM MgCl 2 were added to the suspensions prior to lysing the cells. Cells were lysed by sonication on ice for 10 cycles of 20 sec on 40 sec off, until the lysate was clear and no more DNA was released. The lysate was centrifuged at 13000 × g for 25 minutes at 4°C. The supernatant was decanted and filtered through a 0.45 μm Amicon syringe filter.
The fusion protein was purified with approximately 10 mL Glutathione Superflow Resin (Generon) packed in a free-flow gravity column. The lysate was then added to the column and mixed with the beads. The mixture was left for 10 min to allow binding. The column was washed with 5 column volumes of Lysis Buffer, then with 5 column volumes of Lysis buffer supplemented with 1 M salt, followed with a further wash with 5 column volumes of Lysis buffer. The protein was eluted with 20 mM glutathione (pH 7.0). The GST tag was then cleaved using recombinant HRV-3C protease.
Size exclusion chromatography was performed on Superdex 75 (26/60) column (GE healthcare) attached to an Akta prime system at 4°C. The absorbance of the eluate was monitored at 280 nm throughout. The column was equilibrated with phosphate buffer (40 mM sodium phosphate, 100 mM NaCl, 1 mM DTT, 5% glycerol, pH 7.5). 5 mL of protein (after affinity chromatography) was loaded onto the column using a 5 mL injection loop. Each fraction from within the peak of the UV trace was analysed by SDS-PAGE.

eIF4e (35-216)
pETsumo-eIF4e was transformed in to BL21 (DE3) pLysS Star cells and over-expressed by autoinduction. Overnight cultures were grown in minimal media (Table ESI 1) and 400 μL of this overnight culture was used to inoculate 400 mL auto-induction media (Table ESI 2). The culture was grown for 4 hours at 37°C with aeration, then reduced to 20°C with aeration until no more cell growth was apparent (no more increase in OD). Cells were harvested and resuspended in 15 mL Lysis buffer per 400 mL culture. mM NaCl, 1 mM DTT, 5% glycerol, pH 7.5). 5 mL of protein sample (after affinity chromatography) was loaded onto the column using a 5 mL injection loop. Each fraction from within the peak of the UV trace was analysed by SDS-PAGE.

Ac-SMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQVN-NH 2
The labelled analogue has the same sequence with an additional aminohexanoic acid (Ahx) residue was the N-terminus which was functionalised with fluorescein isothiocyanate (FITC).

Peptide Synthesis
Additional peptides were synthesized by manual or automated Fmoc solid phase peptide synthesis methods on Rink Amide MBHA resin. For each coupling step 5 eq. of the Fmoc-Amino acid in DMF was added to the resin along with 5 eq. of HCTU and 5 eq. of DIPEA in DMF and mixed for 2 hours followed by washing the resin 3 times for 2 minutes with DMF. For the Fmoc deprotection the resin was treated with an excess of 20% Piperidine in DMF 5 times for 2 minutes each followed by washing 5 times with DMF for 2 minutes each.

Acetylation
The resin bound peptide was treated with 10 eq. of acetic anhydride and 10 eq. of DIPEA and mixed overnight.

Fluorescein Labelling
Fmoc-amino hexanoic acid was coupled to the resin bound peptide and deprotected as before and it was then treated with fluorescein isothiocyanate (1.2 eq.) overnight in the minimum volume of 12:7:5 pyridine/DMF/CH 2 Cl 2 . The pyridine was distilled over CaH 2 immediately before use.

Deprotection and Cleavage
The resin was washed with DMF 3 times and DCM 3 times prior to cleavage and deprotection. The peptides were globally deprotected and cleaved from the resin with TFA/thioanisole/water/phenol/EDT (83:5:5:5:2) [Reagent K], concentrated in vacuo, precipitated with ice cold ether, washed 3 times with ice cold ether and dried in vacuo. Peptides were then purified by preparative HPLC and lyophilised.

Determination of the Binding of Labelled HIF-1 Peptide to p300 Protein
Protein was serially diluted into a solution of labelled peptide (80 nM) and the plates incubated for 30 minutes at room temperature. Each experiment was run in triplicate and the fluorescence anisotropy measured using a EnVision 2103 MultiLabel plate reader (Perkin Elmer) with excitation at 480 nm and emission at 535 nm (5 nm bandwidths). The Intensity was calculated for each point using Eq. 1 and used to calculated anisotropy using Eq. 2. From a plot of anisotropy against protein 28 concentration, the minimum and maximum anisotropies were obtained using a logistic sigmoidal fit in OriginPro 8.6.

Competition Assays
Compounds to be tested were serially diluted across a 384 well plate in buffer containing the minimum DMSO (final concentration <5%) before labelled peptide and protein were added sequentially. In parallel, a control experiment was performed in which no labelled peptide was added and the volume made up with additional buffer. Each experiment was performed in triplicate using a EnVision 2103 MultiLabel plate reader (Perkin Elmer) with excitation at 480 nm and emission at 535 nm (5 nm bandwidths) at 25°C. Intensity and anisotropy were calculated as above using Eq. 1 and Eq. 2 respectively. Plots of anisotropy against compound concentration were fitted to a logistic sigmoidal dose response model to determine IC 50 values.

Determination of the Binding of Labelled eIF4G Peptide to eIF4E Protein
Development of a fluorescence anisotropy binding assay for eIF4E/eIF4G was performed as for the HIF-1a/p300 interaction described above.  The assay was validated for identification of competitive inhibitors through competition titration with unlabelled peptide as described for the HIF-1/p300 interaction above.

DOCKING
The protein structure for p300 (PDB ID: 1L8C) was prepared using the protein preparation wizard within Maestro (Schrodinger) and the docking grid prepared by selecting the binding groove of the C-terminal helix of the HIF-1α CTAD using Glide (Schrodinger).
Conformer libraries of compounds were generated using Omega (Openeye Scientific) and prepared for docking using Ligprep (Schrodinger). Docking was performed using Glide (Schrodinger) allowing flexible ligands but penalizing non-planar amide bonds.