An Activity‐Based Probe Targeting Non‐Catalytic, Highly Conserved Amino Acid Residues within Bromodomains

Abstract Bromodomain‐containing proteins are epigenetic modulators involved in a wide range of cellular processes, from recruitment of transcription factors to pathological disruption of gene regulation and cancer development. Since the druggability of these acetyl‐lysine reader domains was established, efforts were made to develop potent and selective inhibitors across the entire family. Here we report the development of a small molecule‐based approach to covalently modify recombinant and endogenous bromodomain‐containing proteins by targeting a conserved lysine and a tyrosine residue in the variable ZA or BC loops. Moreover, the addition of a reporter tag allowed in‐gel visualization and pull‐down of the desired bromodomains.


Chemical Proteomics Experiments
Unless otherwise specified, reagents, solvents, buffers and kits were purchased from Bio-Rad®, Sigma-Aldrich®, Thermofischer®, Lumiprobe® and used without any further purification and/or alteration. The reported procedures were adapted from previously published protocols. 1-2 1.1 In gel visualization of recombinant BRD4(1) using Cy 5.5 IR dye. Sample A: 3.8 µL of a 20 µM solution of Bromotriazine (2) in DMSO were added to 146 µL of a solution of the truncated form of BRD4(1) (1.0 mg/mL) in PBS to a final concentration of 0.5 µM. Sample B: 3.8 µL of a 20 µM solution of BTZ-Non clickable analogue (2b) in DMSO were added to 146 µL of a solution of the truncated form of BRD4(1) (1.0 mg/mL) in PBS to a final concentration of 0.5 µM. Samples A and B were incubated in an Eppendorf Thermomixer TM at 37°C shaking at 300 rpm for 1.5 hours. A 5 µL aliquot was collected from each sample, diluted to 10 µL and stored at -80°C. The remaining samples were then transferred into new tubes in 40 µL aliquots for the click reaction step. 1 µL of a 1.25 mM solution of Cy5.5 azido-dye (a molecule endowed with far red/ near infrared emission) in PBS was added to samples Acetone P, A1 and B1 to a final dye concentration of approximately 25 µM. 1 µL of a 0.5 mM solution of Cy5.5 azido-dye in PBS was added to samples A2 and B2 to a final dye concentration of approximately 10 µM, and 1 µL of a 0.25 mM solution of Cy5.5 azido-dye in PBS was added to samples A3 and B3, to a final dye concentration of approximatively 5 µM. 5 µL of 10% SDS were added and the solution mixed by vortexing before adding 5 µL of the catalyst mix containing 3 parts of TBTA (1.7 mM), 1 part of copper sulfate (50 mM) and 1 part of TCEP (50 mM). The obtained samples were incubated for 1.5 hour at 37°C shaking at 300 rpm. 0.5 µL of a 500 mM solution of EDTA were added and the samples precipitated by the addition of cold acetone (Acetone P) or by chloroform/methanol extraction (A1-A3 and B1-B3). The dried precipitated pellets were resuspended in 30 µL of 10%SDS and run on a polyacrylamide gel. The fluorescent gel was visualized using an Odyssey® CLx IR scanner (Supplemental

Pull down of recombinant BRD4(1) using Biotin-azide.
Sample A: 12.5 µL of a 200 µM solution of Bromotriazine (2) in DMSO were added to 475 µL of a solution of the truncated form of BRD4(1) (0.5 mg/mL) in PBS to a final concentration of 5 µM. Sample B: 12.5 µL of a 200 µM solution of BTZ-Non clickable analogue (2b) in DMSO were added to 475 µL of a solution of the truncated form of BRD4(1) (0.5 mg/mL) in PBS to a final concentration of 5 µM. Sample C was treated as sample A until the click chemistry step. Samples A-C were incubated in an Eppendorf Thermomixer TM at 37°C shaking at 300 rpm for 1.5 hours. The proteins were precipitated by incubating the samples in acetone for 2 hours at -26°C and the obtained pellets re-dissolved in 97.5 µL of PBS and 12.5 µL of 10% SDS via sonication. 2.5 µL of a 50 mM solution of Biotin-azide in PBS were added to Samples A and B, while only PBS was added to sample C. 12.5 µL of the catalyst mix containing 3 parts of TBTA (17 mM), 1 part of copper sulfate (500 mM) and 1 part of TCEP (500 mM) was added to each sample and the reaction incubated for 30 minutes at 37°C shaking at 300 rpm. 5 µL aliquot was removed at this point, 1 µL of Laemmli 4x was added and the aliquots were snap frozen and kept at -80°C (Input Samples to Affinity Precipitation). The remaining samples were precipitated by adding 1 mL of cold acetone to every sample and incubating the resulting suspension at -26°C overnight. The precipitated sediment was spun at 20000 x g for 10 minutes at 4°C, the supernatant was removed by aspiration and 0.5 mL of cold acetone were added. The samples were sonicated to break up and disperse the precipitated proteins and then returned to -80°C for 10 minutes. The supernatant was removed and the obtained pellets dried at the air before being redissolved in 50 µL of 1% SDS in PBS via sonication. The obtained solutions were diluted up by adding 0.5 mL of affinity purification buffer (50 mM HEPES, 100 mM NaCl, 1% NP-40). 20 µL aliquots were removed from each samples, added of 4 µL of Laemmli 4x and kept at -80°C (Efficiency of Precipitation and Resolubilization). 50 µL of a slurry of Streptavidin coated magnetic beads was added to each sample and they obtained suspension shaken at 700 rpm at room temperature for 2 hours. The beads were sedimented using a magnetic tube-holders, the supernatant was removed and 20 µL aliquots were kept at -80°C to be run on the gel as Flow Through After Incubation with Beads. The beads were washed twice (50 mM HEPES, 500 mM NaCl, 1% NP-40 in PBS) and eluted first using 50 µL of a 2 mM solution of free Biotin in 1 % NP-40 in PBS and then by boiling the streptavidin off at 90°C for 5 minutes. The samples were run on a pre-packed 4-12% polyacrylamide gel and stained using Coomassie Brilliant Blue staining solution.
Supplemental Figure 3. Coomassie staining of the gel obtained from the pull down of recombinant BRD4 covalently bond to BTZ(2) using Streptavidin coated magnetic beads. The extra bands appearing at higher molecular weights than the pure recombinant protein (15083 Da) were due to protein aggregation and precipitation induced after the incubation with the click reagents. In fact, LC-MS/MS analysis confirmed that all bands corresponded to recombinant BRD4(1).

Attempted in gel visualization of BRD4 in non transfected K-562 cell lysates using Cy 5.5 IR dye.
Sample A: 5 µL of a 20 µM solution of Bromotriazine (1) in DMSO were added to 195 µL of K-562 cell-lysates (1.0 mg/mL) in LBII (HEPES 50 mM, 10% glycerol, KCl 500 mM, TCEP 1 mM, NP40 1%, Roche® Protease Inhibitor cocktail), to a final concentration of 0.5 µM. Sample B: 5 µL of a 20 µM solution of BTZ-Non clickable analogue (2b) in DMSO were added to 195 µL K-562 cell-lysates (1.0 mg/mL) in LBII, to a final concentration of 0.5 uM. Samples A and B were incubated in an Eppendorf Thermomixer TM at 37°C shaking at 300 rpm for 1.5 hours. A 5 µL aliquot was collected from each sample, diluted to 20 µL and stored at -80°C (Protein before Incubation). 180 µL of both sample A and sample B were then transferred into new tubes for the click reaction step. Addition of 10% SDS was avoided at this point since it induced protein precipitation in LBII. 4 µL of a 0.25 mM solution of Cy5.5 azido-dye in PBS were added to both samples to a final dye concentration of approximately 5 µM. 20 µL of the catalyst mix containing 3 parts of TBTA (1.7 mM), 1 part of copper sulfate (50 mM) and 1 part of TCEP (50 mM) were added and the obtained samples were incubated for 1 hour at 37°C shaking at 300 rpm. 0.9 µL of a 5 mM solution of EDTA were added to half of the samples. Protein precipitation was performed in cold acetone or by chloroform/methanol extraction. The dried precipitated pellets were resuspended in 20 µL of 10%SDS and diluted to 80 µL with PBS. To half of the 15 µL aliquots for the gel, 1 µL of DTT was added and the obtained gel visualized using an Odyssey® CLx IR scanner.
Supplemental Figure 4. LI-COR scan of K-562 cell lysates treated with BTZ (2) and its non clickable analogue 2b. The gel showed selective labelling of samples incubated with the clickable compound, BTZ (2), over those incubated with compound 2b. However, the formation of smears and the evenly spread IR fluorescence indicated non-selective and unspecific binding of BTZ (2) to an extensive portion of the proteome.

Pull down of BRD4 from non-transfected K-562 cell lysates
Three different concentrations of Bromotriazine (2) were incubated with K-562 lysates (5mg/mL) in order to qualitatively and quantitatively study the changes protein pull down with the increasing concentration of probe. Sample A1: 10 µL of a 100 µM solution of Bromotriazine (2) in PBS (0.5% DMSO) were added to 200 µL of K-562 cell lysates (5 mg/mL) in LBII to a final concentration of 5 µM. Sample A2: 10 µL of a 1 mM solution of Bromotriazine (2) in PBS (5% DMSO) were added to 200 µL of K-562 cell lysates (5 mg/mL) in LBII to a final concentration of 50 µM. Sample A3: 10 µL of a 2 mM solution of Bromotriazine (2) in PBS (20% DMSO) were added to 200 µL of K-562 cell lysates (5 mg/mL) in LBII to a final concentration of 100 µM. Control: 10 µL of a 100 µM solution of Bromotriazine (2) in PBS (0.5% DMSO) were added to 200 µL of K-562 cell lysates (5 mg/mL) in LBII to a final concentration of 5 µM. Samples A1-A3 were incubated in an Eppendorf Thermomixer TM at 37°C shaking at 300 rpm for 1.5 hours. 10 µL aliquots were taken for the final gel run (Incubation with Bromotriazine). The lysates were divided into 100 µL aliquots and precipitated by adding cold acetone and incubating the samples for 2 hours at -26°C. The obtained pellets were redissolved in 20 µL of 10% SDS via sonication and diluted to 110 µL with PBS. 5 µL samples were taken from each tube to give a final volume of 10 µL for each concentration (Efficiency of Precipitation and Resolubilization). 2.5 µL of a 50 mM solution of Biotin-azide in PBS and 12.5 µL of the catalyst mix containing 3 parts of TBTA (17 mM), 1 part of copper sulfate (500 mM) and 1 part of TCEP (500 mM) were added to each sample, except from the control sample. The reactions were incubated for 1 hour shaking at 300 rpm, at 37 °C and room temperature respectively. 10 µL aliquots were taken at this point, 1 µL of Laemmli 4x was added and the aliquots snap frozen and kept at -80°C (Input Samples to Affinity Precipitation). The remaining samples were precipitated by adding 1 mL of cold acetone to each sample and incubating the resulting suspension at -26°C overnight. The precipitated sediment was spun at 20000 x g for 10 minutes at 4°C, the supernatant was removed by aspiration and 1 mL of cold acetone was added. Samples were sonicated to break up the pellets and disperse the precipitated proteins, they were then returned to -80°C for 10 minutes. The supernatant was removed and the obtained pellets dried at the air before being redissolved in 10 µL of 10% SDS and 10 10 µL of PBS via sonication. The obtained solutions were diluted up by adding 0.5 mL in affinity purification buffer (50 mM HEPES, 100 mM NaCl, 1% NP-40). 20 µL aliquots were removed from each samples, 4 µL of Laemmli 4x were added and kept at -80°C (Efficiency of 2 nd Precipitation and Resolubilization). 175 µL of a slur of Streptavidin coated magnetic beads in affinity purification buffer was added to each sample and the obtained suspension incubated at 4 °C for 2 hours The beads were sedimented using a magnetic tube-holders, the supernatant was removed and 20 µL aliquots were kept at -80°C to be run on the gel as Flow Through After Incubation with Beads. The beads were washed twice (50 mM HEPES, 500 mM NaCl, 1% NP-40 in PBS) and eluted by boiling the streptavidin off in Laemmli buffer at 90°C for 5 minutes. The samples were run on a pre-packed 4-12% polyacrylamide gel. The proteins were transferred on a membrane and blocked with 5-6% blocking agent in PBS for 15 minutes. The membrane was then incubated with an in house prepared anti-BRD4 (mouse) antibody at 4 °C (1/500), washed twice with PBST and incubated with a secondary antibody bearing an excitable Li-COR sensitive dye. Unfortunately, no band corresponding to full length BRD4 could be detected and even the low molecular weight bands at around 80 KDa, which could be truncated forms of BRD4, seemed to disappear after the click reaction step (Supplemental Figure 5). However, stripping the original membrane and blotting it against an anti-Biotin antibody demonstrated the efficiency of the click reaction. In this case, the amount of pull down on the beads was proportional to the concentration of Bromotriazine (2), despite no characteristic band corresponding to BRD4 could be identified (Supplemental Figure 5).

S6
Western Blot -Anti-BRD4 and Anti-Biotin monoclonal antibodies: Supplemental Figure 5. First attempt to pull down BRD4 from non-transfected K-562 cell lysates using BTZ(2). The top membrane was incubated with anti-BRD4 antibodies and visualised using a secondary antibody bearing an excitable Li-COR sensitive dye. Since no BRD4 could be detected after the click reaction step, the membrane was subsequently stripped and blotted against biotin to investigate the efficiency of the click reaction -bottom membrane.
The proteomics analysis of the protein boiled off the beads, especially at 50 and 100 µM probe (2) concentration highlighted that almost 30% of pulled down proteins were nuclear proteins.

Sample Preparation for mass spectrometry analysis
After incubation of K-562 lysates with 100 µM, 50 µM, or 5µM BTZ (2), a biotin handle was appended to the covalent BTZtarget proteins adducts by CuAAC. Pull down of the covalent complexes was performed on Streptavidin-coated magnetic beads and the enriched extracts were purified by filtering the sample through a Micro Bio-Spin® column (Bio-Rad). For in-solution digestions, trypsin from bovine pancreas (1 g/L in 0.1 HCl, Promega) was reconstituted in 800 L of 50 mM ammonium bicarbonate, and 4 L of this solution were added to 100 L of the 3 M solution of BRD (+ probe). The solution was incubated overnight at 37 ºC. After the digestion was complete, the obtained peptides were purified by Sep-PAK C-18 solid-phase extraction according to the manufacturer's instructions (Waters) and concentrated under vacuum and kept at -20 ºC until analysis.

LC-MS/MS analysis of enriched K-562 cell lysates
Samples were analysed on a liquid chromatography tandem mass spectrometry (LC-MS/MS) essentially as described previously. 3 In brief, resulting peptides were analysed on an Acquity nano UPLC system (Waters) supplemented with a 25 cm C18 column, 1.7 µm particle size (Waters) online coupled to an LTQ Orbitrap Elite (Thermo Scientific). Peptides were eluted by applying a 60 min linear gradient from 5% buffer A (0.1% formic acid in water) to 40% buffer B (0.1% formic acid in acetonitrile) at a flowrate of 250 nl/min (approx. 6000 psi). Full MS scans were performed at a resolution of 30.000. Collision-induced dissociation was performed on the twenty most abundant ions per full MS scan using an isolation width of 1.0 Da. Fragment ions were acquired at a resolution of 7.500. All fragmented precursor ions were actively excluded from repeated MS/MS analysis for 15 s.

Data processing and analysis
Processing of MS Data RAW files were imported into Progenesis QIP (v 3.0.6039.34628), and MS/MS spectra exported as mascot generic files (.mgf) files using the 200 most intense peaks without deconvolution for searching. Datasets were searched in Mascot (v2.5.1) using the following parameters: 10 ppm precursor mass accuracy, 0.05 Da fragment mass accuracy, Oxidation (M), Deamidation (N, Q) and Propionamide (K) as variable modifications, Propionamide (C) as a fixed modification, and two missed cleavage sites. We applied 1% FDR at peptide level (both search engines use a target-decoy method for FDR estimation) and an additional Mascot ion score cut-off of 20 before importing search results into Progenesis, where protein quantification was calculated using the Top3 method. We used Perseus software (v 1.6.0.2) to identify BTZ2 enriched proteins applying a student t test, and p values were corrected for multiple testing using permutation FDR with default settings in Perseus. The data was plotted as a volcano plot ( Figure

Pull down of BRDs from non-transfected THP-1 cell lysates
Optimised procedure for chemical proteomics experiments: THP-1 lysates were used in the proteomics experiments, since they constitutively express significant amounts of both BRD4 and BRD2. 30 mg of lysate was diluted to a final concentration of 4 mg/mL in base buffer (50 mM HEPES, 150 mM NaCl, 15 mM MgCl and 5% Glycerol) and spun down at 18000rcf for 20 minutes at 4 °C. The supernatant was separated from the precipitate, Halt TM cocktail inhibitor and 10% NP40 (to final concentration of 0.4%) were added, together with the appropriate amount of compound 2 (in DMSO) to reach a final concentration of 10, 25 and 100 µM, respectively. The obtained samples were incubated at 4 °C up to 16 hours. 1 mL aliquots were taken from each solution at specific time points: 5 min, 1h, 4h, 8h, and 16h, snap frozen and preserved overnight. After the samples were gently thawed, 10% SDS was added to each tube to a final concentration of 1%SDS and the content of each tube vigorously mixed in order to denature the proteins and expose the alkyne moiety of the covalently bound probes. 25 µL of biotin azide 5M stock in DMSO was added, followed by 25 µL of the catalyst mix, which was previously prepared by adding 15 µL TBTA (1.7 mM), 50 µL of TCEP (50 mM), and 50 µL of copper sulfate (50 mM) to 0.135 mL of a 4:1 t-Butanol:DMSO solution. The obtained light green solutions were incubated at 37 °C for 2 hours in an Eppendorf ThermoMixer®. The protein content was precipitated on cold acetone overnight, centrifuged at 1250 rcf for 10 min at 4 °C, and the obtained pellets reconstituted in 150 µL of 1% SDS in base buffer. The complete re-dissolution of the proteins contained in the pellets was obtained by probe sonication (20 seconds cycles -50% amplitude), after which the concentration of each sample was standardised by Pierce TM 660nm Protein Assay. An adequate amount of lysates, calculated in order to keep the total amount of sample submitted to the following step constant, was added to a slurry containing 50 mg of Agarose beads previously reconstituted in base buffer and 1%SDS. Samples were incubated overnight on a rocking platform at room temperature.
The beads were then loaded on Mobicols and washed twice with 10% NP40 and 0.1% DTT in base buffer, followed by a single wash with 0.1% DTT. The excess buffer was removed from the columns by gentle centrifuge spin and the pull down proteins eluted in 72 µL of 2x LDS and 10 mM DTT by heating the samples at 55 °C for 30 minutes in an Eppendorf ThermoMixer®. 5 µL of a iodoacetamide (IAA) stock solution (200 mg/mL) was added and each sample run on three separate polyacrylamide gel, which were then submitted to silver staining (Supplemental Figure 6), following to the procedure reported in the Pierce TM Silver Stain Kit, and Western Blotting using anti-BRD4 (CSL BRD4 (E2A7X) Rabbit mAb #13440) and anti-BRD2 (CSL BRD2 (D89B4) Rabbit mAb #5848) monoclonal antibodies, respectively. Both WB where developed on photographic film after incubation with HRP-labelled secondary antibody (Supplemental Figures 7-8).

Quantitative Chemical Proteomics on THP-1 cell lysates THP-1 lysate generation
THP-1 lysates were generated as described previously. 4 In brief, THP-1 cell pellets were thawed on ice and resuspended twice the cell pellet volume with ice cold lysis buffer (50 mM HEPES at pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 0.8% NP-40, 1X HALT protease inhibitor (Peirce Biotechnology). The resuspended cell pellet subjected to dounce homogenization and was spun at 800 x g for 10 min at 4 ⁰C. The resulting supernatant (S0.8) was saved, while the pellet was further lysed using via pressure cycling, subjected to salt extraction, and treatment with benzonase. This further processed lysate was combined with the previously saved S0.8 lysate and used as input material for affinity enrichment experiments.

Affinity enrichment and compound competition experiments in THP-1 lysates.
For each affinity enrichment condition, 1 mL of THP-1 lysate (5 mg) was preincubated with either DMSO or 100 µM competition ligand for 16 hr at 4 ⁰C in binding assay buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl2, 0.4% NP-40). The preincubated lysates, were then incubated with either DMSO or 100 µM BTZ for 4 hr at 4 ⁰C. Probe-modified proteins were enriched, digested, subjected to isobaric labeling using TMT tags and sample analysis using nano-LC-MS/MS as previous described. 5 Supplemental Figure 9: Quantitative Proteomics Analysis of Competition Experiments using BTZ (2), its non-clickable analogue (2b), and a known BRD inhibitor (JQ1(+)). THP-1 cell lysates were incubated for 4 hr with 100 µM BTZ (2) at 4°C after preincubation with 100 µM non-clickable analogue (2b) or 100 µM JQ1(+), respectively. The control sample was preincubated with DMSO. BRD2, BRD3, BRD4 and BRDT were neither significantly enriched nor competed and could be detected only after performing targeted MS/MS experiments, indicating that the probe-labeled amount of BET family members was very low. Significant amounts of TMSB4X and HSPE1 were enriched and competed by pre-treatment with both 2b and JQ1(+), thus indicating that these proteins could be potential BTZ (2) off-targets. Dashed lines indicate 50% competition (y-axis) or 2 fold enrichment (x-axis), plotted protein fold changes are median renormalized as described in supplementary Table 1.

Q_SPEC
Number of unique spectra matched to this protein that were used for quantitation (not shared with any other protein sequence in list of identified proteins) LOG10FC_JQ1 Log10 fold change 100 uM JQ1(+) over 1 uM BTZ

Materials and Methods
Cloning, protein expression and purification of bromodomains cDNA encoding reported human bromodomains were cloned, expressed and purified as previously described. 6

Co-purification of BRD9 with bromotriazine (2)
Purified BRD9 (UniProt Q9H8M2, residues 14 -134) was mixed with BTZ (2) in a molar ratio of 1:1.5 M and leave the mixture 3 h at room temperature. A further purification step was carried out using a size exclusion chromatography column in 20 mM Hepes pH 7.5, 350 mM NaCl. The co-eluted peak fraction of the complex was pooled, buffer exchanged in 20 mM Hepes pH 7.5, 100 mM and concentrated up to 15.2 mg/ml.

Crystallization
Aliquots of purified BRD9-BTZ (2) were set up for crystallization using a mosquito® crystallization robot (TTP Labtech, Royston UK). Coarse screens were typically setup onto Greiner 3-well plates using three different drop ratios of precipitant to protein per condition (200+100 nl, 150+150 nl and 100+200 nl). Initial hits were optimized further scaling up the drop sizes. All crystallizations were carried out using the sitting drop vapor diffusion method at 293.15 K. Crystals were grown by mixing 200 nl of the protein with 100 nl of reservoir solution containing 0.1M ammonium citrate pH7, 30% PEG3350. Diffraction quality crystals grew within a few days and were cryo-protected using the well solution supplemented with additional 20 % ethylene glycol and flash frozen in liquid nitrogen.

Data Collection and Structure solution
Crystallographic data was collected at Diamond beamline I03 at a wavelength of 0.9762 Å. Indexing and integration was carried out using XDS 7 and scaling was performed with AIMLESS 8 . Initial phases were calculated by molecular replacement with PHASER 9 using the apo BRD9 structure (PDB IDs 3HME). Initial models were built by ARP/wARP 10 followed by manual building in COOT 11 . Refinement was carried out in REFMAC5 12 . GRADE (global phasing) 13 was used to generate compound coordinates and cif files. Thermal motions from individual monomers of the octamer within the asymmetric unit were analyzed using TLSIN (REFMAC), and hydrogen atoms were included in late refinement cycles. All model validations were carried out using MolProbity 14 . Data collection and refinement statistics can be found in Supplemental Table 1. The model and structure factors have been deposited with PDB accession code: 5O4B.pdb

Crystallographic data collection and refinement statistics
Supplementary

Docking Data
Docking experiments were performed using the Molsoft L.L.C.® ICM-Pro software and the crystal structure of BRD4(1) (PDB code: 4OGI).

BTZ (2) and BRD4(1)
The poses generated by docking BTZ (2) in the binding pocket of BRD4(1) and the corresponding scores are shown in Supplementary Figure 9 and Table 3

Synthetic Procedures and NMR Spectra
Solvents were used as supplied from Fisher Scientific®, Acros Organics® or Sigma Aldrich® without further purification. Where mixtures of solvents are specified, the stated ratios are volume:volume. Unless otherwise indicated, all aqueous solutions used were saturated. Reagents were used directly as supplied by major chemical suppliers. Column chromatography was carried out using a Biotage Isolera TM Prime and pre-packed columns for flash chromatography. Analytical thin-layer chromatography was carried out on Sigma-Aldrich® silica gel or aluminium oxide on TLA aluminium foils with fluorescent indicator at 254 nm. Visualization was carried out under ultra-violet irradiation (254 nm). NMR spectra were recorded on a Bruker AV400 (1H: 400 MHz, 13C: 101 MHz). Chemical shifts are quoted in ppm, based on appearance rather than interpretation, and are referenced to the residual non-deuterated solvent peak. High resolution mass spectra were recorded on a QTof interfaced with a LC or a Rapidfire TM mass spectrometer. The previously reported synthesis of bromosporine 15 was optimised and adapted to produce compounds 2 and 2b, as shown in Scheme A and B.

3,6-Dichloropyridazin-4-amine (4):
In a sealed vial, 1.0 g (5.4 mmol) of 3,4,6-trichloropyridazine (3) were suspended in 10 mL of conc. ammonium hydroxide and heated up to 90 °C for 1 hour. The reaction was cooled down to room temperature, 10 mL of water were added and the precipitate was collected by filtration to give title compound as an off white solid (0.58 g, 65% yield).

6-Chloro-3-hydrazinylpyridazin-4-amine (5):
In a sealed vial, 1.0 g (6.1 mmol) of 3,6dichloropyridazin-4-amine (4) were suspended in 10 mL of hydrazine monohydrate and heated up to 90 °C for 3 hours. The reaction was cooled down to room temperature, poured on ice, and the resulting precipitate was collected by filtration to give title compound as a pale yellow solid (0.48 g, 49% yield).

6-Chloro-3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-amine (6):
In a round bottom flask, 3.0 g (19.0 mmol) of 6-chloro-3-hydrazinylpyridazin-4-amine (5) were suspended in 25 mL of conc. acetic acid and heated up to 90 °C for 3 hours. The reaction was cooled down to room temperature, poured on ice, and the resulting precipitate was collected by filtration. A solution of sat. sodium carbonate was added to the obtained solid and the resulting suspension stirred for half an hour at room temperature. The insoluble solid was filtered off to give title compound as an off white solid (2.7 g, 77% yield).
But-3-yn-1-yl carbonochloridate (11): Triphosgene (10.6 g, 35.7 mmol) was dissolved in 20 mL of anhydrous THF and the solution cooled down to 0°C. But-3-yn-1-ol (5.4 mL, 71.3 mmol) was diluted in 10 mL of dry THF and added dropwise over 2h at 0°C. The resultant mixture was warmed up to room temperature and stirred overnight. Anhydrous pyridine (8.6 mL, 107 mmol) was added dropwise to the solution over 1h at 0°C and then the reaction was warmed to room temperature and stirred for a further hour. Precipitated pyridinium hydrochloride salt was removed by filtration and the THF evaporated under vacuum to give title compound as an orange oil (5.8 g, quantitative yield).

Kinetic Experiments:
Experimental conditions: 100 µM probe was incubated with 1 µM bromodomain in 20 mM HEPES and 200 mM NaCl at 37 ºC. At given time points (0,5,10,20,30,60,90 and 120 minutes), the solution was sampled (75 µL), purified through a quick solid phase extraction on C4 cartridge and analysed by Rapidfire 360 TM MS interfaced with a QTOF. Blank experiments were Values are reported as absolute area of each mass peak.

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Area values were converted into concentrations (µM) by assuming a direct proportionality between deconvoluted areas and concentrations. The average of deconvoluted areas registered during the control experiment is referred as the concentration of BRD at t=0 ([A0] = 1 µM) as it doesn't change during the experiment and it takes into account experimental errors.