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A public-private partnership that supports the discovery of new medicines through open access research.
Bromosporine (BSP) This compound is available from Tocris, Sigma and Cayman Chemical For any inquiries please contact proberequests@thesgc.org.group newOverview Bromodomains (BRDs) are protein interaction modules that read epigenetic marks recognizing ε-N-lysine acetylation motifs. The conserved BRD fold contains a deep, largely hydrophobic acetyllysine binding site, an attractive pocket for the development of small, pharmaceutically active molecules. BRDs have an important role in the targeting of chromatin-modifying enzymes to specific sites, including methyltransferases, HATs and transcription factors and regulate diverse biological processes from cell proliferation and differentiation to energy homeostasis and neurological processes. Co-crystal structure of Bromosporine with BRD4(1) Structure of Bromosporine Proteins that contain BRDs have been implicated in the development of a large variety of diseases, including various cancers, inflammatory diseases and neurological diseases and the therapeutic potential of bromodomain inhibition has been shown in several of these diseases, such as HIV, cancer and inflammation. Bromosporine is a broad spectrum inhibitor for bromodomains and as such will be very useful in elucidating further biological roles of reader domains as well as a tool for the validation of functional assays. Properties ethyl N-[6-(3-methanesulfonamido-4-methylphenyl)-3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl]carbamate Click here to download SDF file Physical and chemical properties Molecular weight 404.44 Molecular formula C17H20N6O4S IUPAC Name ethyl N-[6-(3-methanesulfonamido-4-methylphenyl)-3-methyl-[1,2,4]triazolo[4,3-b]pyridazin-8-yl]carbamate clogP 0.78 PSA 105.64 Storage 2-8°C as powder. NB making aliquots rather than freeze-thawing is recommended Dissolution Soluble in DMSO at least up to 50mM SMILES: CC1=C(NS(C)(=O)=O)C=C(C2=NN3C(C(NC(OCC)=O)=C2)=NN=C3C)C=C1 InChI: InChI=1S/C17H20N6O4S/c1-5-27-17(24)18-15-9-14(21-23-11(3)19-20-16(15)23)12-7-6-10(2)13(8-12)22-28(4,25)26/h6-9,22H,5H2,1-4H3,(H,18,24) InChIKey: UYBRROMMFMPJAN-UHFFFAOYSA-N Selectivity Profile 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. Bromosporine 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. 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. The temperature dependence of the fluorescence during the protein denaturation process was approximated by the equation (displayed below) where ΔuG(T) is the difference in unfolding free energy between the folded and unfolded state, R is the gas constant and yF and yU are the fluorescence intensity of the probe in the presence of completely folded and unfolded protein respectively. The baselines of the denatured and native states were approximated by a linear fit. The observed temperature shifts, ΔTmobs, were recorded as the difference between the transition midpoints of sample and reference wells containing protein without ligand in the same plate and determined by non-linear least squares fit. Selectivity Bromodomain Thermal melt 10 µM ΔTm BRD2(1) 4.4 BRD2(2) 5.9 BRD3(1) 5.3 BRD3(2) 5.9 BRD4(1) 6.9 BRD4(2) 6.2 BRDT(1) 7.3 BRDT(2) 5.2 CECR2 8.3 PB1(5) 0.4 TAF1(1) 1.3 TAF1(2) 5.2 TAF1L(1) 0.9 TAF1L(2) 0.9 BAZ2A 1.2 TIF1α 0.4 ATAD2 -0.2 BRD9 3.9 CREBBP 3.4 Cell-based Assay Data Potency in Cells BRD4 Accelerated FRAP recovery at 1 µM CREBBP Accelerated FRAP recovery at 1 µM TIF1α Inactive at 10 µM BAZ2A Inactive at 10 µM SMARCA2 Inactive at 10 µM Bromosporine shows moderate cytotoxicity in HeLa cells at 18 µM Synthetic SchemesScheme 1 Scheme 2 References Promiscuous targeting of bromodomains by bromosporine identifies BET proteins as master regulators of primary transcription response in leukemia Sarah Picaud, Katharina Leonards, Jean-Philippe Lambert, Oliver Dovey, Christopher Wells, Oleg Fedorov, Octovia Monteiro, Takao Fujisawa, Chen-Yi Wang, Hannah Lingard, Cynthia Tallant, Nikzad Nikbin, Lucie Guetzoyan, Richard Ingham, Steven V. Ley, Paul Brennan, Susanne Muller, Anastasia Samsonova, Anne-Claude Gingras, Juerg Schwaller, George Vassiliou, Stefan Knapp and Panagis Filippakopoulos. Science Advances 12 Oct 2016: Vol. 2, no. 10, e1600760. DOI: 10.1126/sciadv.1600760