The probe is available from Sigma and Cayman Chemical.
| Probe |
 |
OF-1 |
A chemical probe for the bromodomains of the BRPF (BRomodomain and PHD Finger containing) family of proteins (BRPF1/2/3) has been discovered by the SGC.
BRPF1, BRPF2 (BRD1) and BRPF3 are scaffolding proteins, assembling HAT complexes of the MOZ/MORF family (MOZ, Ybf2/Sas3, Sas2, and Tip60) (1). These MYST complexes have a tetrameric core containing BRPF, the tumour suppressor ING and Eaf6/EPC (enhancer of polycomb)-related scaffold subunits. MYST complexes play crucial roles in DNA repair, recombination, and replication as well as in transcription activation (2,3). MOZ is frequently translocated in AML (acute myeloid leukemia) and is required for HSC proliferation (4).
Two BRPF1 isoforms (isoform A and B) can be generated by alternative splicing. In contrast to BRPF1B, the isoform A harbours a residue insertion in the ZA-loop that prevents binding to acetylated histone peptides (5).
Phylogenetic tree of bromodomains and detailed view at the BRPF family.
OF-1 has been shown to bind to BRPF1B with a KD of 100 nM (ITC), to BRPF2 with a KD of 500 nM (ITC) and to BRPF3 with a KD of 2.4 µM (ITC). Selectivity against other BRDs is very good, in general >100-fold. The closest off-target effects are against BRD4 (39-fold selectivity) and TIF1α (50% inhibition at 20 µM in the alpha screen). OF-1 increases thermal stability in the cellular thermal shift assay (CETSA) of full length BRPF1B at 1 µM and also demonstrates accelerated FRAP recovery at 5 µM in the BRPF2 FRAP assay. It shows modest general cytotoxicity.
| Bromodomain | Kd/nM (ITC) | IC50/nM (Alpha Screen) | TM Shift °C |
| BRPF1B | 100 | 270 | 6.5±0.4 |
| BRPF2 (BRD1) | 500 | 2200 | 3.1±0.3 |
| BRPF3 | 2400 | ND | 3.3±0.2 |
| BRD4 (1) | 4000 | >10,000 | 2.0±0.2 |
OF-1 is a chemical probe with potent binding affinity for BRPF1 (KD of 100nM), BRPF2 (KD of 500 nM) and BRPF3 (KD of 2400 nM) as determined by isothermal titration calorimetry (ITC). Alpha screen biochemical assay confirmed OF-1 as a potent inhibitor of BRPF1 (IC50 of 270 nM).
OF-1 induced significant ΔTm shifts within the BRPF family. Weak interactions (2.1 °C) were also observed for BRD4(1), however alpha screen did not reveal strong interactions of OF-1 with BRD4 (IC50 of >10000 nM) and a KD of 4000nM for BRD4(1) reveals 39-fold selectivity when compared to BRPF1B isoform.
OF-1 shows no significant inhibition of protein kinases (<20% inhibition at 10 μM for 40 kinases screened).
We recommend to use the BRPF inhibitor NI-57 in parallel to confirm the result. Please do not apply OF-1 higher than 5 μM to avoid effects on BRD4 and verify the result by using NI-57.
OF-1 increases thermal stability in the CETSA of full length BRPF1B at 1 µM.
In a NanoBRETTM assay, BRPF1B, but not BRPF1A isoform shows dose-dependent displacement from histone H3.3, with IC50 of 0.08 μM and the FRAP assay reveals strong inhibition of BRPF2 (BRD1) at 5 µM concentration of OF-1.
OF-1 attenuates RANKL/MCSF induced differentiation of primary human monocytes into osteoclasts.
| Probe |
|
OF-1 |
| Physical and chemical properties | |
|---|---|
| Molecular weight | 440.31 |
| Molecular formula | C17H18BrN3O4S |
| IUPAC name | 4-bromo-N-(6-methoxy-1,3-dimethyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)-2-methylbenzenesulfonamide |
| clogP | 2.55 |
| PSA | 78.95 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 4 |
| No. of hydrogen bond acceptors | 4 |
| No. of hydrogen bond donors | 1 |
| Storage | Stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | Soluble in DMSO |
Temperature Shift Assay
Selectivity screening of chemical probe OF-1 determined by temperature shift assay.
The temperature shifts mapped onto the phylogenetic tree using red circles corresponding to ΔTm as indicated in the figure.
CESTA
Thermal-stability of BRPF1A/B determined by CESTA at 1 µM OF-1.
NanoBRET
Dose-dependent displacement of BRPF1A/B from histone H3.3 following treatment with OF-1.
FRAP Assay
Half-times of fluorescence recovery (t1/2) after photo bleaching measured for BRPF2 (BRD1) after treatment with OF-1 at different concentrations with or without SAHA.
TRAP staining for osteoclasts and measurement of pits depth.
Work on this probe has been published in 'Selective targeting of Bromodomain-PHD fingers family (BRPF) bromodomains impairs osteoclast differentiation'.
Isothermal Titration Calorimetry (ITC)
All calorimetric experiments were performed on a VP-ITC micro-calorimeter (MicroCalTM, LLC Northampton, MA). Protein solutions were buffer exchanged by gel filtration or dialysis into buffer (20 mM Hepes pH 7.5, 150 mM NaCl, and 0.5 mM tris (2-carboxyethyl) phosphine (TCEP). All measurements were carried out at 288.15 K. All injections were performed using an initial injection of 2 µL followed by injections of 8 µL. The data were analysed with the MicroCal ORIGIN software package employing a single binding site model. The first data point was excluded from the analysis.
Temperature shift assay
Thermal melting experiments were carried out using a Stratagene Mx3005p Real Time PCR machine (Agilent Technologies). OF-1 was added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000 as described (6).
AlphaScreen Assay
Assays were performed as described previously with minor modifications (7). All reagents were diluted in 25 mM HEPES, 100 mM NaCl, 0.1 % BSA, pH 7.4 supplemented with 0.05 % CHAPS and allowed to equilibrate to room temperature prior to addition to plates. An 11-point 1:2.0 serial dilutions of the ligands was prepared on lowvolume 384-well plates (ProxiPlateTM-384 Plus, PerkinElmer, USA), using LabCyte Eco liquid handler. Plates filled with 5 µL of the assay buffer followed by 7 µL of biotinylated peptide [H-YSGRGKacGGKacGLGKacGGAKacRHRK(Biotin)-OH for BRD1, BRD4, BRPF1B and BRPF3 or YQTARKSTGGK(ac)APRKQLATKAK(biotin)-OH for TIF1α] and Histagged protein to achieve final assay concentrations of 25-100 nM depending on the dose-response curve for each individual protein. Plates were sealed and incubated for a further 30 minutes, before the addition of 8 µM of the mixture of streptavidin-coated donor beads (12.5 µg/mL) and nickel chelate acceptor beads (12.5 µg/mL) under low light conditions. Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation/570 emission filter set. IC50 values were calculated in Prism 5 (GraphPad Software, USA) after normalization against corresponding DMSO controls.
Fluorescence Recovery After Photobleaching (FRAP) Assay
FRAP studies were performed using U20S cells expressing full-length BRPF2 (BRD1). Six hours after transfection 2.5 µM SAHA (to increase global histone acetylation) was added and cells were treated with 1 µM or 5 µM of OF-1 1 hour before imaging and half recovery times from the fluorescence signal of the bleached U2OS nuclei were plotted.
NanoBRET
U2OS cells were co-transfected with Histone H3.3-HaloTag and NanoLuc-BRPF1. Twenty hours post-transfection cells were collected, washed with PBS, and exchanged into media containing phenol red-free DMEM and 4% FBS in the absence (control sample) or the presence (experimental sample) of 100 nM NanoBRET 618 fluorescent ligand (Promega). Cells were then treated with an increasing dose of OF-1. Five minutes prior to reading, NanoBRET furimazine substrate (Promega) was added to both control and experimental samples and plates were read on a CLARIOstar (BMG) equipped with 450/80 nm bandpass and 610 nm longpass filters with a 0.5 sec reading setting. A corrected BRET ratio was calculated and is defined as the ratio of the emission at 610 nm/450 nm for experimental samples (i.e. those treated with NanoBRET fluorescent ligand) subtracted by and the emission at 610 nm/450 nm for control samples (not treated with NanoBRET fluorescent ligand). BRET ratios are expressed as milliBRET units (mBU), where 1 mBU corresponds to the corrected BRET ratio multiplied by 1000. Relative IC50 values were estimated by non-linear regression analysis of (log) concentration of each inhibitor versus milliBRET ratios (GraphPad Prism).
Human osteoclast differentiation
Primary human peripheral blood mononuclear cells (PBMCs) were collected from a Histopaque generated buffy coat after gradient centrifugation at 20 min and 500g, brakes off. The CD14+ monocyte fraction was obtained by on-column CD14+-MACS bead isolation, washed twice with MACS buffer, and seeded at a density of 50 000 c/mL in αMEM/10%FCS supplemented with 25 ng/mL MCSF. After 6 days at 37 °C, 5% CO2 treatment with OF-1 with and without 50 ng/mL RANKL was started. Media were changed with fresh compound every 3−4 days. After 14−21 days, cells were fixed and stained for TRAP.
Bone Resorption Assays.
PBMCs were isolated and seeded onto self-cut dentine slices from ivory. After 14 days of differentiation, cells were removed from dentine slices. Dentine pits were imaged with confocal microscopy.
Toronto, Canada & New York City, USA — The Structural Genomics Consortium (SGC) and CHDI Foundation have entered into a unique open-access research collaboration to discover and characterize new drug targets for Huntington’s disease (HD) using structural and chemical biology.
SGC707 is available from Cayman Chemical and Sigma and from Tocris
XY1 (the negative control of SGC 707) is available from Tocris and from Cayman Chemical.
| Probe | Negative control | |
 |
|  |
SGC707 (IC50=31 nM) |
| XY1 (IC50>100,000 nM) |
A collaboration between the SGC, China Novartis Institutes for Biomedical Research, and the center for Integrative Chemical Biology and Drug Discovery (CICBDD) at the University of North Carolina has resulted in the discovery of SGC707, a chemical probe for PRMT3, a protein which catalyzes the asymmetrical di-methylation of arginine residues. SGC707 is a potent allosteric inhibitor of PRMT3 (IC50 = 50 nM) with >100-fold selectivity over other methyltransferases, and other non-epigenetic targets. SGC707 has been shown to bind to PRMT3 with KD of 50 nM (ITC), and inhibits the methylation of histones in cells with IC50 value below 1 µM.
| Probe | Negative control | |
|
| |
SGC707 (IC50=31 nM) |
| XY1 (IC50>100,000 nM) |
Click here to download SDF file
| Physical and chemical properties | |
|---|---|
| Molecular weight | 298.1430 |
| Molecular formula | C16H18N4O2 |
| IUPAC name | 2-((3-aza-bicyclo[4.4.0]deca-1,3,5,7,9-pentaen-8-ylamino)-formylamino)-1-(pyrrolidin-1-yl)-ethanone |
| logP | 1.8 |
| PSA | 59.9 A |
(C) Effect of SGC707 on the activity of 27 protein methyltransferases as well as DNMT1, DNMT3A-3L, DNMT3B-3L and BCDIN3D (an RNA-methyltransferase) were assessed.
(D) IC50 values were determined at saturating concentration of peptide substrate (1.5 µM) and SAM concentrations equal to 0.25, 0.5, 1, 2, 4, 8, 10 and 12x of the Km for SAM. No change in IC50 values at varying SAM concentrations was consistent with a noncompetitive pattern of inhibition with respect to SAM. (E) To determine the competition with peptide, the SAM concentration was kept at saturation (70 µM) and IC50 values were determined at different peptide concentrations (0.5, 1, 2, 4, 8, 12, 16, and 20 ×Km). A similar noncompetitive pattern was observed for SGC707 with respect to peptide confirming the allosteric mode of inhibition.
Western blot analysis of H4R3me2a levels. HEK293 cells were co-transfected with FLAG tagged PRMT3 (WT) or its catalytically dead mutant E335Q (Mut) and treated with different concentrations of SGC707, as indicated. Total cell lysates were collected 24 h post inhibitor treatment and analysed for H4R3me2a levels. The total levels of exogenous histone H4 and overexpressed PRMT3 were determined with anti-GFP, anti-H4 and anti-FLAG antibodies, respectively
Kaniskan, H. Ü., Szewczyk, M. M., Yu, Z., Eram, M. S., Yang, X., Schmidt, K., Luo, X., Dai, M., He, F., Zang, I., Lin, Y., Kennedy, S., Li, F., Dobrovetsky, E., Dong, A., Smil, D., Min, S.-J., Landon, M., Lin-Jones, J., Huang, X.-P., Roth, B. L., Schapira, M., Atadja, P., Barsyte-Lovejoy, D., Arrowsmith, C. H., Brown, P. J., Zhao, K., Jin, J. and Vedadi, M. (2015), A Potent, Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 3 (PRMT3).
Angew. Chem.. doi: 10.1002/ange.201412154
The Group led by Professor Chas Bountra, Chief Scientist of SGC Oxford, has been awarded the Royal Society of Chemistry's Rita and John Cornforth Award 2014 for "... world leading collaborative research across the disciplines of structural biology, medicinal chemistry, chemical biology and enzymology towards understanding and exploiting the potential of epigenetics as a target family for future drug discovery".
This probe is available from Cayman Chemical , Tocris and Sigma-Aldrich.
The control is available from Cayman Chemical.
| Probe | Negative control | |
 |
|  |
OICR-9429 |
| OICR-0547 |
A collaboration between the SGC and The Drug Discovery Program at the Ontario Institute for Cancer Research (OICR) has resulted in the discovery of OICR-9429, a chemical probe for WDR5, a component of several chromatin regulatory complexes including the MLL1 (Mixed Lineage Leukemia 1) complex. OICR-9429 is a potent antagonist of the interaction of WDR5 with peptide regions of MLL and Histone 3 with >100-fold selectivity over other chromatin “reader” domains, methyltransferases, and other non-epigenetic targets. OICR-9429 has been shown to bind to WDR5 with KD values of 24 nM (Biacore) and 52 nM (ITC). OICR-9429 also disrupts the interaction of WDR5 with MLL1 and RbBP5 in cells with IC50 values below 1 µM.
A closely-related compound, OICR-0547, does not show WDR5 antagonism, and can therefore be used as a negative control in phenotypic assays.
| Probe | Negative control | |
|
| |
OICR-9429 |
| OICR-0547 |
| Physical and chemical properties for OICR-9429 | |
| Molecular weight | 555.2 |
| Molecular formula | C29H32F3N5O3 |
| IUPAC name | 5-((2-(4-methyl-piperazin-1-yl)-5-(3-((morpholin-4-yl)-methyl)-phenyl)-phenylamino)-formyl)-4-(trifluoro-methyl)-1,2-dihydro-pyridin-2-one |
| MollogP | 3.382 |
| PSA | 65.34 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 8 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 2 |
| Physical and chemical properties for OICR-0547 (Negative Control) | |
| Molecular weight | 542.2 |
| Molecular formula | C28H29F3N4O4 |
| IUPAC name | 5-((2-(morpholin-4-yl)-5-(3-((morpholin-4-yl)-methyl)-phenyl)-phenylamino)-formyl)-4-(trifluoro-methyl)-1,2-dihydro-pyridin-2-one |
| MollogP | 3.331 |
| PSA | 69.77 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 8 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 2 |
Pharmacological targeting of the Wdr5-MLL interaction in C/EBPa N-terminal leukemia ,Florian Grebien, Masoud Vedadi, Matthäus Getlik, Roberto Giambruno, Amit Grover, Roberto Avellino, Anna Skucha, Sarah Vittori, Ekaterina Kuznetsova, David Smil, Dalia Barsyte-Lovejoy, Fengling Li, Gennadiy Poda, Matthieu Schapira, Hong Wu, Aiping Dong, Guillermo Senisterra, Alexey Stukalov, Kilian V M Huber,Andreas Schönegger, Richard Marcellus, Martin Bilban, Christoph Bock, Peter J Brown, Johannes Zuber, Keiryn L Bennett, Rima Al-awar, Ruud Delwel, Claus Nerlov, Cheryl H Arrowsmith & Giulio Superti-Furga. Nature Chemical Biology. 10.1038/nchembio.1859
Main features
The probe is available from Tocris , Cayman Chemical and Sigma
| Probe |
 |
BAZ2-ICR |
Biology of the BAZ2 Bromodomains
BAZ2A/B belong to a family of ubiquitously expressed bromodomain containing proteins. Proteins of the BAZ (bromodomain adjacent zinc finger) family are characterized by a carboxy-terminal bromodomain adjacent to a PHD finger and a WACZ motif. In addition four other conserved motifs are typically found in the N-terminus of BAZ family members, namely the LH motif (a leucine-rich helical domain), the ZB2 motif (conserved between ZK783.4 and BAZ2 proteins) and the BAZ 1 and BAZ 2 motifs (conserved between all BAZ proteins, Acf, and ZK783.4).
Little is known about the biological function of BAZ2B but it has been suggested to regulate nucleosome mobilization by the ATP-dependent chromatin remodeling factor ISWI. Interaction of BAZ2B with ISWI is mediated by the BAZ1 motif [1]. The BAZ2B locus has also been identified as associated with sudden cardiac death [2]. The related BAZ2A (also called TIP5) protein plays a central role in the NoRC (nucleolar remodeling complex). BAZ2A is essential for heterochromatin formation leading to transcriptional silencing of certain rRNAs [3].
BAZ2-ICR
A collaboration between the SGC and Swen Hoelder’s group at the Institute of Cancer Research, London (ICR) has resulted in the discovery of BAZ2-ICR, a chemical probe for BAZ2A/B [4]. This is the second probe identified against this target, GSK2801, has previously been reported [5].
Co-crystal structure
The co-crystal structure of BAZ2-ICR and BAZ2B has been solved (pdb id 4XUB)
Potency Against Target Family
Selectivity
The inhibitor was screened at 10 μM concentration against 47 bromodomains using a temperature shift assay. The screened targets are labelled on the phylogenetic tree; targets that have not been screened are shown in grey. Temperature shifts are represented as spheres as indicated in the figure.
electivity Beyond Target Family
BAZ2-ICR (10 µm) displayed no activity against a panel of 55 receptors and ion channels
Cellular Activity
BAZ2-ICR shows accelerated FRAP recovery at 1 µM in the BAZ2A FRAP assay
| BAZ2-ICR |
|
4-(4-(1-methyl-1H-pyrazol-3-yl)-1-(2-(1-methyl-1H-pyrazol-4-yl)ethyl)-1H-imidazol-5-yl)benzonitrile |
An inactive, negative control, compound for BAZ2-ICR has yet to be identified
| Physical and chemical properties | |
|---|---|
| Molecular weight | 357.42 |
| Molecular formula | C20H19N7 |
| IUPAC name | 4-(4-(1-methyl-1H-pyrazol-3-yl)-1-(2-(1-methyl-1H-pyrazol-4-yl)ethyl)-1H-imidazol-5-yl)benzonitrile |
| logP | 2.54 |
| PSA | 70.59 |
| No. of chiral centres | 0 |
| No. of rotatable bonds | 5 |
| No. of hydrogen bond acceptors | 7 |
| No. of hydrogen bond donors | 0 |
| Storage | Stable as solid in the dark at -20°C. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | 25 mM in D2O |
Potency against Target
BAZ2-ICR has been shown by ITC to bind to BAZ2A with a KD of 109 nM (ITC) and to BAZ2B with a KD of 170 nM. The strong negative binding enthalpy possibly due to aromatic stacking interaction of pyrozole/phenyl
Selectivity Within Target family
BAZ2-ICR showed significant thermal shifts of 5.2 and 3.8 °C for BAZ2A and BAZ2B respectively. BAZ2-ICR did not show significant thermal shifts against all other bromodomains, except for Cat Eye syndrome chromosome region, candidate 2 (CECR2), where a smaller but significant shift (ΔTm: 2.0 °C) was observed. The Kd of BAZ2-ICR binding to CERC2 was determined to be 1.55 μM by ITC, giving a 15-fold and 10-fold selectivity over BAZ2A and BAZ2B respectively.
Click the images to enlarge them
AlphaScreen Assay
To confirm selectivity over BRD4, BAZ2-ICR assay was studied in a BRD4 AlphaScreen assay, where it did not show significant inhibition up to 50 μM, translating in greater than 100-fold window.
Selectivity Beyond Target Family
BAZ2-ICR (10 µm) displayed no activity against a panel of 55 receptors and ion channels
FRAP
To investigate whether BAZ2-ICR can displace BAZ2 bromodomains from chromatin in living cells, we performed a fluorescence recovery after photobleaching (FRAP) assay utilizing GFP-tagged BAZ2A full length protein transfected into human osteosarcoma cells (U2OS). Mutant BAZ2A (N1873F) that does not bind acetylated lysine containing peptides and therefore mirrors the behaviour of inhibitor bound BAZ2A was used as a control. The histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) was used to increase overall levels of histone acetylation, resulting in a sufficient assay window to enable the measurement of differences in recovery time and demonstrating the acetylation dependence of the FRAP experiments. BAZ2-ICR at 1 μM reduced the recovery time of the wt construct to a level similar to the mutant, confirming that BAZ2-ICR inhibits BAZ2A in cells. Attempts to clone full length BAZ2B for this assay were unsuccessful.
Click the images to enlarge them
To confirm selectivity in the cellular setting, a similar FRAP assay for BRD4 selectivity was performed. Cells were transfected with GFP-tagged BAZ2A or BRD4 and treated with 1 μM PFI-1 (a selective probe for BET Bromodomains) or BAZ2-ICR. Light bars depict assay with SAHA addition, and dark bars depict assays without SAHA addition (2.5 μM). Fluorescence recovery (t½) expressed as a percentage of the relevant wild-type control cells without inhibitor; the dotted line demarks the point equivalent to 100% of the relevant wild-type control cells without inhibitor. Error bars depict the standard error of the mean. Bars marked with an asterisk indicate a significant difference from controls (P < 0.05).
The physicochemical and mouse pharmacokinetic properties of BAZ2-ICR were assessed. BAZ2-ICR showed very high solubility (25 mM in D2O), a measured log D of 1.05, high stability in mouse microsomes, and permeation in the CaCo-2 model and thus a suitable profile for oral and intravenous gavage. We therefore performed a full mouse pharmacokinetic experiment. In agreement with the in vitro data, BAZ2-ICR showed 70% bioavailability and moderate clearance (∼50% of mouse liver blood flow) and volume of distribution, suggesting that BAZ2-ICR is suitable for modulating BAZ2A and BAZ2B in vivo.
3 mice were dosed IV and 3 mice PO. Key parameters and the curves are shown for each animal.
The co-crystal structure of BAZ2-ICR and BAZ2B has been solved (pdb id 4XUB)
The key interactions are illustrated below.
Isothermal Titration Calorimetry
Experiments were carried out on a VP-ITC titration microcalorimeter from MicroCalTM, LLC (Northampton, MA), at 15 °C while stirring at 295 rpm, in ITC buffer (50 mM HEPES, 150 mM NaCl). All titrations were conducted using an initial injection of 2 µl followed by 34 identical injections of 8 µl with a duration of 16 sec/injection and a spacing of 250 sec between injections. The heat of dilution was determined by independent titrations (protein into buffer) and was subtracted from the experimental data. MicroCalTM Origin software was used to calculate enthalpies of binding (ΔH) and binding constants (KB). Thermodynamic parameters were calculated (ΔG = ΔH - TΔS = -RTlnKB, where ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding respectively). In all cases a single binding site model was employed.
Differential Scanning Fluorimetry (DSF)
Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 µM in 20 µl volume. Compounds were added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000. Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3 °C per minute from 25 °C to 96 °C and fluorescence readings were taken at each interval. Data was analysed as previously reported [7]
CEREP assay
BAZ2-ICR (10 µm) was screened against a panel of 55 ligand receptors, ion channels and transports using an established and widely utilized commercial assay platform (ExpresSProfile; CEREP, Paris, FRANCE).
Fluorescence Recovery After Photobleaching (FRAP) Assay
FRAP studies were performed using U20S cells expressing full-length BAZ2A or BRD4 protein fused with an N-terminal eGFP as previously described [4]. Six hours after transfection 2.5uM SAHA (to increase global histone acetylation) was added and BAZ2-ICR was added 1 hour before imaging, which was carried out 24 hours after transfection. Percent inhibition was calculated between the DMSO treated (0%) and N1873F expressing mutant (100%)
Oxford, UK and Fremont, US - March 3, 2014 – The Structural Genomics Consortium (SGC) and DiscoveRx Corporation announced the publication of findings demonstrating that several clinical kinase inhibitors also potently inhibit diverse bromodomain epigenetic reader proteins.
The probe GSK-LSD-1 (hydrochloride) is available from Cayman Chemical, Sigma and Tocris.
| Probe |
 |
GSK-LSD1 |
GSK-LSD1 (trans-racemic)
MW 216.3
FW 289.2 (di-HCl)
Lysine specific demethylase 1 (LSD1) is a histone demethylase found in various transcriptional co-repressor complexes. LSD1 is involved in ES cell differentiation, hematopoiesis, and has been described as having a role in Acute Myeloid Leukemia (AML). GlaxoSmithKline has developed an irreversible, mechanism based inhibitor of LSD1, GSK-LSD1, and has made this available as a chemical probe as part of the SGC epigenetics initiative. GSK-LSD1 inhibits LSD1 with an IC50 of 16 nM and is > 1000 fold selective over other closely related FAD utilizing enzymes (i.e. LSD2, MAO-A, MAO-B). GSK-LSD1 induces gene expression changes in cancer cell lines (average EC50 < 5 nM) and inhibits cancer cell line growth (average EC50 < 5 nM).
LSD-1 at 10 µM showed no activity against 55 human recombinant receptors in the CEREP ExpresSProfile selectivity/specificity profile (GPCR, transporters, ion channels), except 5-HT1A (49% inhibition) 5-HT transporter (74% inhibition) and dopamine transporter (39 % inhibition).
| Probe |
|
GSK-LSD1 |
| Physical and chemical properties for GSK-LSD1 | |
| Molecular weight | 216.2 |
| Molecular formula | C14H20N2 |
| IUPAC name | N-(2-phenyl-cyclopropyl)-piperidin-4-amine |
| MollogP | 2.639 |
| PSA | 23.66 |
| No. of chiral centres | 2 |
| No. of rotatable bonds | 3 |
| No. of hydrogen bond acceptors | 2 |
| No. of hydrogen bond donors | 2 |
Toronto, December 18, 2013 – Bayer Inc. is pleased to announce that Bayer HealthCare has joined the Structural Genomics Consortium (SGC), a not-for-profit, public-private partnership with active research facilities at the Universities of Toronto and Oxford, UK. Bayer will support funding the consortium to accelerate precompetitive drug research in the areas of protein sciences and epigenetics. Furthermore, Bayer will provide a subset of its compound library for screening to the SGC and will also conduct the chemical work to identify probes.
This probe is available from Cayman Chemical, Sigma and Tocris
| Probe | Negative control | |
 |
|  |
PFI-3 |
| PFI-3oMet |
| In vitro Potency | |
| Assay | Kd/IC50 (nM) |
| SMARCA4 (ITC) | 89 |
| PB1(5) (ITC) | 48 |
SMARCA4 (SWI/SNF related, Matrix associated, Actin dependent Regulator of Chromatin, subfamily A, member 4), also known as BRG1 (Brahma-Related Gene 1), is part of the SWI/SNF (SWItch/Sucrose NonFermentable) family of proteins. Multiple transcript variants encoding different isoforms have been found for this gene in normal tissue as well as in cancer. SMARCA4 and the related protein SMARCA2 (BRM, BRahMa) contain a bromo and helicase domain and both proteins are central components of the large ATP-dependent SNF/SWI chromatin remodelling complex, which plays a key role in chromatin remodelling and transcription control. PB1 (PolyBromo 1) can also be part of the SWI/SNF complex.
Loss of function of SMARCA4 and components of SWI/SNF has been linked to cancer development suggesting a tumour suppressor function for BRG1. SMARCA4 has also been shown to interact with the breast cancer gene BRCA1 (BReast CAncer 1, early onset), as well as to regulate the expression of the tumorigenic protein CD44.
We have developed an inhibitor, PFI-3, against the SMARCA2/4 and PB1(5) bromodomains.
|
| (2E)-1-(2-hydroxyphenyl)-3-[(1R,4R)-5-(pyridin-2-yl)-2,5-diazabicyclo[2.2.1]heptan-2-yl]prop-2-en-1-one Click here to download SDF file |
| Physical and chemical properties | |
|---|---|
| Molecular weight | 321.4 |
| Molecular formula | C 19H 19N 3O 2 |
| IUPAC name | (2E)-1-(2-hydroxyphenyl)-3-[(1R,4R)-5-(pyridin-2-yl)-2,5-diazabicyclo[2.2.1]heptan-2-yl]prop-2-en-1-one |
| logP (ChemBioDraw Ultra) | 2.39 |
| PSA (ChemBioDraw Ultra) | 56.14 |
| Storage | -20°C as powder. NB making aliquots rather than freeze-thawing is recommended |
| Dissolution | Soluble in DMSO at least up to 10mM |
| Stability* at 37°C | t 1/2 > 15 days |
| Stability* at 20°C | t 1/2 > 200 days |
*as measured by LCMS
PFI-3 only shows a significant Tm shift with PB1(5) and SMARCA2/4. No interaction was observed with PB1(2-4), and there was no cross-reactivity in a kinase panel of 36 kinases.
| Selectivity | |
|---|---|
| Bromodomains | |
| Target | Tm shift °C @ 10 µM |
| SMARCA2 | 6.4 |
| SMARCA4 | 5.1 |
| PB1 1st Bromodomain | -0.5 |
| PB1 2nd Bromodomain | 1.1 |
| PB1 3rd Bromodomain | 0.9 |
| PB1 5th Bromodomain | 7.5 |
| PB1 6th Bromodomain | 0.4 |
| PCAF | 0.5 |
| BRD4 1st Bromodomain | 0.3 |
| BRD4 2nd Bromodomain | 0.4 |
| CREBBP | -0.0 |
| BRD1 | 0.0 |
| TIF1alpha | 0.7 |
| TRIM2B | 0.3 |
| TAF1L 1st Bromodomain | 0.2 |
| TAF1L 2nd Bromodomain | 0.3 |
| Other proteins | |
| Invitrogen kinase panel | No activity |
| Selectivity | |
|---|---|
| SMARCA2 (Frap Assay) | Accelerated FRAP recovery at 1 uM |
| SMARCA2 (Frap Assay) * | Accelerated FRAP recovery at 1 uM |
| SMARCA2 (Frap Assay) # | Accelerated FRAP recovery at 1 uM |
*24 hour compound incubation on cells
^1 hour compound incubation on cells preceded by 24 hour pre-incubation of the compound in medium
Selective targeting of the BRG/PB1 bromodomains impairs embryonic and trophoblast stem cell maintenance. Oleg Fedorov, Josefina Castex, Cynthia Tallant, Dafydd R. Owen, Sarah Martin, Matteo Aldeghi, Octovia Monteiro, Panagis Filippakopoulos, Sarah Picaud, John D. Trzupek, Brian S. Gerstenberger, Chas Bountra, Dominica Willmann, Christopher Wells, Martin Philpott, Catherine Rogers, Philip C. Biggin, Paul E. Brennan, Mark E. Bunnage, Roland Schüle, Thomas Günther, Stefan Knapp, Susanne Müller
Science Advances 13 Nov 2015:
Vol. 1, no. 10, e1500723
DOI: 10.1126/sciadv.1500723
