| # | Aladdin ID | Assay Type | Description | Organism | Compounds | Reference | BAO Format | Source | |
|---|---|---|---|---|---|---|---|---|---|
| 1. | ALA3889117 | B | Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 μs read window). Data were normalized based on a positive (2 μM I-BET) and negative (DMSO) controls and IC50values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations. | 32 | ALA3886951 | single protein format | BindingDB Database | ||
| 2. | ALA3889118 | B | Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 μs read window). Data were normalized based on a positive (2 μM I-BET) and negative (DMSO) controls and IC50values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations. | 32 | ALA3886951 | single protein format | BindingDB Database | ||
| 3. | ALA3889119 | B | Amplified Luminescent Proximity Homogeneous Assay (ALPHA): Binding of the bromodomain BRD4-I to an acetylated histone H4 peptide was measured using a bead-based Amplified Luminescent Proximity Homogeneous Assay (ALPHA). The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-I was expressed and purified from Escherichia coli as an N-terminal His6-tagged protein. Nickel-Chelate ALPHA acceptor beads (Perkin Elmer) were used to specifically bind BRD4-1 and ALPHA streptavidin donor beads (Perkin Elmer) were used because they specifically recognized the biotinylated H4 peptide. Binding of BRD4-1 to the peptide resulted in proximity of the donor and acceptor beads which leads to an increase in ALPHA signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in ALPHA signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO, 200 nM H4 peptide and 15 nM of BRD4-1 protein. After an assay reaction time of 60 minutes at 25° C., binding was measured with 20 ug/ml streptavidin donor beads and 20 ug/ml nickle-chelate acceptor beads. ALPHA signal was detected on an Envision plate reader (Ex: 320 nm; Em: 570 nm; Ex time: 180 ms). Data were normalized based on a positive (2 uM I-BET) and negative (DMSO) controls and IC50 values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations. | 27 | ALA3886951 | single protein format | BindingDB Database | ||
| 4. | ALA4332474 | B | Inhibition of biotin-labelled tetra-acetylated Histone H4 peptide binding to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD4 bromodomain1 after 60 mins by HTRF assay | Homo sapiens | 17 | ALA4330118 | single protein format | Scientific Literature | |
| 5. | ALA4332475 | B | Inhibition of biotin-labelled tetra-acetylated Histone H4 peptide binding to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD4 bromodomain2 after 60 mins by HTRF assay | Homo sapiens | 17 | ALA4330118 | single protein format | Scientific Literature | |
| 6. | ALA4332476 | F | Cytotoxicity against human MT4 cells assessed as reduction in cell viability after 5 days by CellTiter-Glo assay | Homo sapiens | 18 | ALA4330118 | cell-based format | Scientific Literature | |
| 7. | ALA4332477 | A | Effect on MYC transcription in human MM1S cells measured after 6 hrs | Homo sapiens | 1 | ALA4330118 | cell-based format | Scientific Literature | |
| 8. | ALA4332478 | B | Binding affinity to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD2 bromodomain1 assessed as change in melting temperature at 30 uM measured after 30 mins by SYPRO orange dye based differential scanning fluorimetry | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 9. | ALA4332479 | B | Binding affinity to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD2 bromodomain2 assessed as change in melting temperature at 30 uM measured after 30 mins by SYPRO orange dye based differential scanning fluorimetry | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 10. | ALA4332480 | B | Binding affinity to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD3 bromodomain1 assessed as change in melting temperature at 30 uM measured after 30 mins by SYPRO orange dye based differential scanning fluorimetry | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 11. | ALA4332481 | B | Binding affinity to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD3 bromodomain2 assessed as change in melting temperature at 30 uM measured after 30 mins by SYPRO orange dye based differential scanning fluorimetry | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 12. | ALA4332482 | B | Binding affinity to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD4 bromodomain1 assessed as change in melting temperature at 30 uM measured after 30 mins by SYPRO orange dye based differential scanning fluorimetry | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 13. | ALA4332483 | B | Binding affinity to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD4 bromodomain2 assessed as change in melting temperature at 30 uM measured after 30 mins by SYPRO orange dye based differential scanning fluorimetry | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 14. | ALA4332484 | B | Binding affinity to recombinant human N-terminal TEV-cleavable hexa-histidine tagged CREBBP assessed as change in melting temperature at 30 uM measured after 30 mins by SYPRO orange dye based differential scanning fluorimetry | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 15. | ALA4332485 | B | Binding affinity to recombinant human N-terminal TEV-cleavable hexa-histidine tagged SMARCA4 assessed as change in melting temperature at 30 uM measured after 30 mins by SYPRO orange dye based differential scanning fluorimetry | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 16. | ALA4332486 | B | Inhibition of Cy5-linked JQ1 probe binding to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD4 bromodomain1 after 60 mins by HTRF assay | Homo sapiens | 2 | ALA4330118 | single protein format | Scientific Literature | |
| 17. | ALA4332487 | B | Inhibition of Cy5-linked JQ1 probe binding to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD4 bromodomain2 after 60 mins by HTRF assay | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 18. | ALA4332488 | B | Inhibition of tetra-acetylated Histone H4 peptide binding to recombinant human N-terminal TEV-cleavable hexa-histidine tagged BRD4 bromodomain1 after 60 mins by ALPHA screen assay | Homo sapiens | 3 | ALA4330118 | single protein format | Scientific Literature | |
| 19. | ALA4332489 | B | Binding affinity to BRD4 in human MM1S cells assessed as inhibition of protein binding to IgH super enhancer after 6 hrs by chromatin immunoprecipitation assay | Homo sapiens | 1 | ALA4330118 | cell-based format | Scientific Literature | |
| 20. | ALA4332490 | B | Inhibition of BRD4 in human MM1S cells assessed as reduction in MYC transcription measured after 6 hrs | Homo sapiens | 1 | ALA4330118 | cell-based format | Scientific Literature |
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