Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4

The Bromo- and Extra-Terminal (BET) proteins BRD2, BRD3, and BRD4 play important roles in transcriptional regulation, epigenetics, and cancer and are the targets of pan-BET selective bromodomain inhibitor JQ1. However, the lack of intra-BET selectivity limits the scope of current inhibitors as probes for target validation and could lead to unwanted side effects or toxicity in a therapeutic setting. We designed Proteolysis Targeted Chimeras (PROTACs) that tether JQ1 to a ligand for the E3 ubiquitin ligase VHL, aimed at triggering the intracellular destruction of BET proteins. Compound MZ1 potently and rapidly induces reversible, long-lasting, and unexpectedly selective removal of BRD4 over BRD2 and BRD3. The activity of MZ1 is dependent on binding to VHL but is achieved at a sufficiently low concentration not to induce stabilization of HIF-1α. Gene expression profiles of selected cancer-related genes responsive to JQ1 reveal distinct and more limited transcriptional responses induced by MZ1, consistent with selective suppression of BRD4. Our discovery opens up new opportunities to elucidate the cellular phenotypes and therapeutic implications associated with selective targeting of BRD4.

A: compound VHL-1 bound to the von-Hippel-Lindau protein (pdb-code 4W9H); B: compound VHL-2 bound to the von-Hippel-Lindau protein (pdb-code 4W9K); C: JQ1 bound to the first bromodomain of BRD4 (pdb-code 3XMF). The red circles show the solvent exposed parts of the molecules chosen for connection.        HeLa cells were treated with 100 nM of MZ1, VHL-1′, or JQ1, or 1 µM of JQ1 or 0.01% DMSO vehicle control (Veh.) for (A) 12 hours or (B) 24 hours. Quantitative PCR was performed to analyze relative gene expression level of treated HeLa cells using target specific primers. Gene expression levels relative to GAPDH were normalized to control treatment. The data shown represent the mean ± SEM (n= 3, technical replicates) of one experiment. Statistical significance compared to the control was determined with two-tailed t tests: *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant. mRNA expression of BRD2, BRD3 and BRD4 were selectively suppressed upon transfection of their respective siRNA. HeLa cells were transfected with siRNA targeting individual BRD2, BRD3 or BRD4 or with negative control siRNA and were harvested after 48 hours. Quantitative PCR was performed to analyze relative gene expression level of treated HeLa cells using target specific primers. Gene expression levels relative to GAPDH were normalized to control treatment. The data shown represent the mean ± SEM (n= 3, technical replicates) of one experiment. Statistical significance compared to the control was determined with two-tailed t tests: *P < 0.05; **P < 0.01; ***P < 0.001; n.s. not significant.

Synthetic procedures
The following compounds were prepared according to literature procedures : 4 and 5, 1 PEG linkers 6 and 7. 2

General procedure for Boc-deprotection:
The N-Boc-protected compound was dissolved in dichloromethane (10 ml/1 mmol). Trifluoroacetic acid (10 ml/1 mmol) was added and the reaction mixture stirred at room temperature for 2 h. The solvent was removed under reduced pressure. For three times dichloromethane (5 ml /1 mmol) was added and then the solvent again removed in vacuum to remove residual trifluoroacetic acid.
x Hyp 10 i trans 11 ii trans 12 i cis x Hyp 4 i trans 5 ii trans 9 i cis S13 General procedure for linker coupling: The amine (10 -12) (1 mmol, 1 eq.) was added to a solution of PEG-linker (6, 7) (1.2 mmol, 1.2 eq.) in DCM (40 ml). HATU (570 mg, 1.5 mmol, 1.5 eq.) was added and the pH adjusted to >9 by addition of DIPEA (700 μl, 4 mmol, 4 eq.). After stirring for 4 h at 25 °C the reaction mixture was extracted with water. The organic phase was dried over Magnesium sulfate and evaporated to dryness. The crude product was purified by flash column chromatography using a gradient of 0%-6% of Methanol in Dichloromethane.
General procedure for PROTAC formation: Azides 13 -16 (50 μmol) were dissolved in methanol (5 ml). Catalytic amount of palladium on Charcoal (10 wt%) was added and the reaction mixture stirred under an atmosphere of hydrogen for 3 h at 25 °C. The reaction mixture was filtered through a plug of celite and the resulting solution evaporated to dryness to obtain the desired amine.
The resulting amines (45 μmol, 1.1 eq.) and 17 (16.0 mg, 40 μmol, 1 eq.) were dissolved in DCM (2 ml). HATU (22.8 mg, 60.0 μmol, 1.5 eq.) was added and the pH adjusted to >9 by adding DIPEA (41.9 μl, 240 μmol, 4 eq. was removed in vacuum. Purification of the crude was achieved by preparative HPLC as described in the general information.     . The bacteria was harvested the next day by centrifugation (8000 rpm for 10 minutes at 6 °C, JLA 8.1000 rotor on a Beckman Coulter Avanti J-20 XP centrifuge) and frozen at -20 °C as pellets for storage. Pellets of cells expressing His 6 -tagged proteins were resuspended in lysis buffer (50 mM HEPES pH 7.5 at 25 °C, 150 mM NaCl, 40 mM Imidazole and 2 mM β-mercaptoethanol). One tablet of Complete Protease Inhibitor Cocktail (Roche) was added to the resuspension and cells were lysed using a French Press at 4 °C. Following a 20 min incubation period at room temperature with 10 μg/mL DNaseI and 10 mM MgCl2, the cell debris was removed by centrifugation, 20,000 x g at 4 °C. The lysate was purified via immobilized metal ion affinity chromatography on a His Trap HP 5mL Ni sepharose column (GE Healthcare Life Sciences) on an ÄKTApure system (GE Healthcare). His 6 -tagged protein was eluted using a linear gradient to 250 mM imidazole in the same buffer. After Ni purification, the pooled elution fractions were concentrated to a volume of 4 mL and further purified by size exclusion chromatography on a Superdex 75 16/60 Hiload gel filtration column (GE Healthcare) on an ÄKTApure system using the following buffer: 20 mM HEPES pH 7.5, 150 mM NaCl. Samples were monitored by SDS-polyacrylamide gel electrophoresis to verify purity. Pure protein was then flash frozen with liquid nitrogen and stored at -80 °C. The mass and purity of the proteins were subsequently verified by mass spectrometry.

Isothermal Titration Calorimetry (ITC):
ITC experiments were carried out at an ITC 200 instrument from MicroCal TM with a concentration in the measuring cell of 15 μM and a syringe concentration of 150 μM. Experiments were conducted as PROTAC compound into protein titrations, except in the case of MZ3 where protein was titrated into PROTAC. BET protein experiments were conducted in a buffer containing 20 mM HEPES with 100 mM NaCl at pH 7.5 and a temperature of 30 °C. VBC protein experiments were carried out in a buffer containing 20 mM Bis-Tris, 150 mM NaCl and 2 mM Dithiothreitol (DTT) at pH 7 and a temperature of 25 °C.

Tissue culture
HeLa and U2OS cells were cultured in DMEM supplemented with 10 % FBS, 1 % L-glutamine and 100U/ml of penicillin/streptomycin. Cells were maintained for no more than 30 passages at 37 °C and 5 % CO 2 .

Cell treatment Small interfering RNA
For siRNA inhibition studies, cells were plated in six-well plates and were grown to 50-60% confluence. Cells were transfected with siRNA targeting BRD2, BRD3 or BRD4 or negative control siRNA at a final concentration of 12 nM in the presence of lipofectamine reagent. After transfection, cells were cultured for another 24 hours for treatment with compound or harvested after 48 hours for gene expression study.

Single time point treatment
For treatment experiments cells were transferred in 6-well plates with 500 000 cells per well in 2 ml media. 12 h after settling 200 μl of media was removed and then replaced by a 10 fold concentrated compound solution in media. The final DMSO concentration was 0.01 % v/v.

Time course experiments
For time dependent treatment cells were transferred in 6-well plates with 300 000 cells per well in 2 ml media. For treatment 200 μl of media was removed and then replaced by a 10 fold concentrated compound solution in media. Treatment was conducted at given time points prior to harvest.

Protein recovery experiment
Cells were transferred in 6-well plates with 300 000 cells per well in 2 ml media. For treatment 200 μl of media was removed and then replaced by a 10 fold concentrated compound solution in media. 4 h after treatment the media was aspirated and replaced by fresh media without treatment. Cells were harvested at given time points.

Western blotting
For protein extracts the dishes were placed on ice. The media was aspirated and the tissue layer washed twice with ice cold PBS. 120 μl of RIPA-buffer containing Protease inhibitor was added and the cells detached from the surface with a cell scraper. All PCR reactions were performed using the Bio-Rad CFX96 Touch Real-Time PCR system and the amplifications were done using the Quanta PerfeCTa® SYBR® Green FastMix for iQ (cat. # 95071). The thermal cycling conditions were composed of 95°C for 10 min, 45 cycles at 95°C for 10s and 60°C for 30s followed by a ramping temperature step to 95°C for melt-curve analysis.
The experiments were carried out in triplicate for each data point. The data was analysed using CFX Manager software from Bio-Rad and the relative quantification in gene expression was determined by normalising to the control gene GAPDH.

Fluorescence microscopy:
U2OS cells transiently expressing GFP-tagged BRD4 were prepared. Plasmid pcDNA5/FRT/TO-GFP containing full-length wild-type BRD4 is obtained as described in previous study. 3 U2OS cells were plated onto a glass bottom microwell dish (MatTek, P35G-1.5-14-C) in 2.5 mL medium and were grown to 50-60% confluence. Then the cells were transfected with 4 μg of plasmid DNA in the presence of FuGene 6 Transfection Reagent (Promega, E2691). Twelve hours after S27 transfection, medium was removed in exchange of fresh medium supplemented with 0.5 µg/mL tetracycline to induce GFP-BRD4 expression. After 18 hours, medium was removed again in exchange of fresh medium without tetracycline. After 6 hours, compound MZ1 or cis-MZ1 were added to the plate. Immediately after the treatment, fluorescence given out from individual cells plate were observed on a DeltaVision Elite imaging system with excitation at 480 nm and emission at 525 nm. Images of individual cells were made at regular time interval to observe changes in fluorescence over time.

Legends to Supporting Information videos:
Supporting video a: BRD4 depletion by MZ1 monitored by live fluorescence imaging.
U2OS cells transfected with GFP-BRD4 were treated with 5 μM of MZ1 over a time course of 4 hours. GFP-BRD4 degradation was followed by live fluorescence imaging using a DeltaVision Elite Imaging system. The pictures taken every 2 minutes were combined to a 10 second time lapse video.

Supporting video b: Intracellular amount of GFP-BRD4 does not change in the presence of cisMZ1.
U2OS cells transfected with GFP-BRD4 were treated with 5 μM of cisMZ1 over a time course of 4 hours. Live fluorescence imaging using a DeltaVision Elite Imaging system showed no reduction in fluorescence. The pictures taken every 2 minutes were combined to a 10 second time lapse video.