ChEMBL ligand: CHEMBL1957266
bromodomain containing 1/Bromodomain-containing protein 1 in Human [ChEMBL: CHEMBL2176774] [GtoPdb: 2724] [UniProtKB: O95696] There should be some charts here, you may need to enable JavaScript!
bromodomain containing 2/Bromodomain-containing protein 2 in Human [ChEMBL: CHEMBL1293289] [GtoPdb: 1944] [UniProtKB: P25440] There should be some charts here, you may need to enable JavaScript!
bromodomain containing 3/Bromodomain-containing protein 3 in Human [ChEMBL: CHEMBL1795186] [GtoPdb: 2725] [UniProtKB: Q15059] There should be some charts here, you may need to enable JavaScript!
bromodomain containing 4/Bromodomain-containing protein 4 in Human [ChEMBL: CHEMBL1163125] [GtoPdb: 1945] [UniProtKB: O60885] bromodomain containing 4/Bromodomain-containing protein 4 in Mouse [ChEMBL: CHEMBL3085619] [GtoPdb: 1945] [UniProtKB: Q9ESU6] There should be some charts here, you may need to enable JavaScript!
bromodomain containing 9/Bromodomain-containing protein 9 in Human [ChEMBL: CHEMBL3108640] [GtoPdb: 2728] [UniProtKB: Q9H8M2] There should be some charts here, you may need to enable JavaScript!
bromodomain testis associated/Bromodomain testis-specific protein in Human [ChEMBL: CHEMBL1795185] [GtoPdb: 2729] [UniProtKB: Q58F21] bromodomain testis associated in Mouse [GtoPdb: 2729] [UniProtKB: Q91Y44] There should be some charts here, you may need to enable JavaScript!
C-C motif chemokine 2 in Human [ChEMBL: CHEMBL1649052] [UniProtKB: P13500] There should be some charts here, you may need to enable JavaScript!
CREB binding lysine acetyltransferase/CREB-binding protein in Human [ChEMBL: CHEMBL5747] [GtoPdb: 2734] [UniProtKB: Q92793] There should be some charts here, you may need to enable JavaScript!
Delta and Notch-like epidermal growth factor-related receptor in Human [ChEMBL: CHEMBL5291567] [UniProtKB: Q8NFT8] There should be some charts here, you may need to enable JavaScript!
EP300 lysine acetyltransferase/Histone acetyltransferase p300 in Human [ChEMBL: CHEMBL3784] [GtoPdb: 2735] [UniProtKB: Q09472] There should be some charts here, you may need to enable JavaScript!
phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta/Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform in Human [ChEMBL: CHEMBL3130] [GtoPdb: 2155] [UniProtKB: O00329] There should be some charts here, you may need to enable JavaScript!
phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma/Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform in Human [ChEMBL: CHEMBL3267] [GtoPdb: 2156] [UniProtKB: P48736] There should be some charts here, you may need to enable JavaScript!
Janus kinase 2/Tyrosine-protein kinase JAK2 in Human [ChEMBL: CHEMBL2971] [GtoPdb: 2048] [UniProtKB: O60674] There should be some charts here, you may need to enable JavaScript!

DB	Assay description	Assay Type	Standard value	Standard parameter	Original value	Original units	Original parameter	Reference
bromodomain containing 1/Bromodomain-containing protein 1 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL2176774] [GtoPdb: 2724] [UniProtKB: O95696]
ChEMBL	Inhibition of recombinant BRD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	4.35	pIC50	44500	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
bromodomain containing 2/Bromodomain-containing protein 2 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL1293289] [GtoPdb: 1944] [UniProtKB: P25440]
ChEMBL	Inhibition of human 6x-His-tagged BRD2 bromodomain 1 expressed in Escherichia coli	B	6.89	pKd	130	nM	Kd	J Med Chem (2018) 61: 9316-9334 [PMID:30253095]
ChEMBL	Affinity Biochemical interaction (Isothermal titration calorimetry (ITC)) EUB0000330a BRD2	B	6.89	pKd	128.4	nM	Kd	Affinity Biochemical Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Binding affinity to BRD2-BD1 by isothermal titration calorimetry	B	6.89	pKd	128.4	nM	Kd	Bioorg Med Chem (2012) 20: 1887-1892 [PMID:22316554]
ChEMBL	Binding affinity to His-tagged BRD2 bromodomain 2 (unknown origin) by SPR assay	B	6.94	pKd	116	nM	Kd	Bioorg Med Chem (2018) 26: 25-36 [PMID:29170024]
ChEMBL	Binding affinity to BRD2 bromodomain 2 (348 to 455 residues) (unknown origin) by ITC	B	6.95	pKd	112.14	nM	Kd	Bioorg Med Chem (2018) 26: 25-36 [PMID:29170024]
ChEMBL	Binding affinity to recombinant BRD2 BD1 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.11	pKd	78	nM	Kd	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Binding affinity to recombinant BRD2 BD2 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.28	pKd	52	nM	Kd	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Binding affinity to BRD2 D1 (unknown origin) assessed as dissociation constant by isothermal titration calorimetry	B	7.47	pKd	34	nM	Kd	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Binding affinity to biotinylated BRD2 BD1 (72 to 205 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells by bio-layer interferometry method	B	7.83	pKd	14.7	nM	Kd	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Binding affinity to BRD2 BD1 (unknown origin) by thermal denaturation assay	B	7.96	pKd	11	nM	Kd	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Binding affinity to BRD2 BD2 (unknown origin) by thermal denaturation assay	B	8.05	pKd	9	nM	Kd	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Binding affinity to biotinylated BRD2 BD2 (349 to 460 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells by bio-layer interferometry method	B	8.21	pKd	6.2	nM	Kd	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Displacement of FAM-labeled ZBA248 from BRD2 BD1 (72 to 205 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells after 30 mins by fluorescence polarization assay	B	7.88	pKi	13.2	nM	Ki	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD2 bromodomain 1 (72 to 205 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.88	pKi	13.2	nM	Ki	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Displacement of FAM-labeled ZBA248 from BRD2 BD2 (349 to 460 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells after 30 mins by fluorescence polarization assay	B	7.9	pKi	12.5	nM	Ki	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD2 bromodomain 2 (349 to 460 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.9	pKi	12.5	nM	Ki	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Inhibition of BRD2 BD1/2 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.01	pIC50	982	nM	IC50	Eur J Med Chem (2024) 271: 116444-116444 [PMID:38691889]
ChEMBL	Binding affinity to human partial length BRD2 BD1 (K71 to N194 residues) expressed in bacterial expression system using H4K5acIC8acK12acK16ac-biotinylated peptide as substrate preincubated for 30 mins followed by substrate addition by AlphaScreen assay	B	6.09	pIC50	820	nM	IC50	J Med Chem (2023) 66: 913-933 [PMID:36577036]
ChEMBL	Inhibition of recombinant BRD2 BD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.13	pIC50	733	nM	IC50	J Med Chem (2023) 66: 1239-1253 [PMID:36622852]
ChEMBL	Inhibition of recombinant BRD2 BD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.41	pIC50	392	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
ChEMBL	Inhibition of BRD2 BD1 (unknown origin) incubated for 2 hrs by TR-FRET assay	B	6.73	pIC50	185	nM	IC50	J Med Chem (2021) 64: 18025-18053 [PMID:34908415]
ChEMBL	Inhibition of recombinant human BRD2 bromodomain 2 (342 to 460 residues) using biotin labeled peptide substrate preincubated for 15 mins followed by substrate addition measured after 1 hr by TR-FRET assay	B	6.77	pIC50	170	nM	IC50	Bioorg Med Chem (2018) 26: 25-36 [PMID:29170024]
ChEMBL	Inhibition of BRD2 bromodomain 1 (unknown origin) by Alphascreen based method	B	6.81	pIC50	155	nM	IC50	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Inhibition of human recombinant His-tagged BRD2 BD1 domain expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells by AlphaScreen assay	B	6.81	pIC50	155	nM	IC50	J Med Chem (2020) 63: 3678-3700 [PMID:32153186]
ChEMBL	Inhibition of BRD2 D1 (unknown origin) by competitive fluorescence anisotropy assay	B	6.85	pIC50	140	nM	IC50	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Inhibition of BRD2 D2 (unknown origin) by competitive fluorescence anisotropy assay	B	6.92	pIC50	120	nM	IC50	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Inhibition of recombinant BRD2 BD2 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.95	pIC50	112	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
ChEMBL	Inhibition of BRD2 BD1 (unknown origin) by Alphascreen assay	B	6.96	pIC50	110	nM	IC50	J Med Chem (2024) 67: 18606-18628 [PMID:39356741]
ChEMBL	Inhibition of BRD2 BD1 (unknown origin) after 1 hr by TR-FRET assay	B	7.11	pIC50	78	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Binding affinity BRD2 BD1 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.11	pIC50	77	nM	IC50	J Med Chem (2022) 65: 2388-2408 [PMID:34982556]
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	7.2	pIC50	63	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	Binding affinity BRD2 BD2 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.27	pIC50	54	nM	IC50	J Med Chem (2022) 65: 2388-2408 [PMID:34982556]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD2 bromodomain 2 (349 to 460 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.27	pIC50	53.3	nM	IC50	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Inhibition of BRD2 BD2 (unknown origin) after 1 hr by TR-FRET assay	B	7.28	pIC50	52	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Inhibition of BRD2 BD2 (unknown origin) by Alphascreen assay	B	7.3	pIC50	50	nM	IC50	J Med Chem (2024) 67: 18606-18628 [PMID:39356741]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD2 bromodomain 1 (72 to 205 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.44	pIC50	36.1	nM	IC50	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Inhibition of BRD2 BD2 (unknown origin) incubated for 2 hrs by TR-FRET assay	B	7.49	pIC50	32	nM	IC50	J Med Chem (2021) 64: 18025-18053 [PMID:34908415]
ChEMBL	Inhibition of BRD2 BD1 (unknown origin) preincubated for 15 mins followed by peptide addition and measured after 60 mins by TR-FRET assay	B	7.54	pIC50	29	nM	IC50	Eur J Med Chem (2020) 208: 112780-112780 [PMID:32883643]
ChEMBL	Inhibition of BRD2 BD1 (unknown origin) by TR-FRET assay	B	7.55	pIC50	28	nM	IC50	Eur J Med Chem (2023) 251: 115246-115246 [PMID:36898329]
ChEMBL	Inhibition of BRD2-BD1/2 (unknown origin) by HTRF analysis	B	7.59	pIC50	26	nM	IC50	J Med Chem (2023) 66: 2646-2662 [PMID:36774555]
ChEMBL	Inhibition of BRD2 (unknown origin) by TR-FRET assay	B	7.72	pIC50	19	nM	IC50	Medchemcomm (2019) 10: 974-984 [PMID:31303996]
ChEMBL	Inhibition of BRD2 bromodomain 2 (unknown origin) by Alphascreen based method	B	7.77	pIC50	17	nM	IC50	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Inhibition of human recombinant His-tagged BRD2 BD2 domain expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells by AlphaScreen assay	B	7.77	pIC50	17	nM	IC50	J Med Chem (2020) 63: 3678-3700 [PMID:32153186]
ChEMBL	Binding affinity to human partial length BRD2 BD2 (E348to D455 residues) expressed in bacterial expression system using H4K5acIC8acK12acK16ac-biotinylated peptide as substrate preincubated for 30 mins followed by substrate addition by AlphaScreen assay	B	8.03	pIC50	9.25	nM	IC50	J Med Chem (2023) 66: 913-933 [PMID:36577036]
ChEMBL	Inhibition of BRD2 BD2 (unknown origin) by TR-FRET assay	B	8.05	pIC50	9	nM	IC50	Eur J Med Chem (2023) 251: 115246-115246 [PMID:36898329]
ChEMBL	Inhibition of BRD2 BD2 (unknown origin) preincubated for 15 mins followed by peptide addition and measured after 60 mins by TR-FRET assay	B	8.17	pIC50	6.7	nM	IC50	Eur J Med Chem (2020) 208: 112780-112780 [PMID:32883643]
ChEMBL	Affinity Phenotypic Cellular interaction (Cell Proliferation (Cell TiterGlo assay Promega in NMC 11060 cells)) EUB0000330a BRD2	F	8.4	pIC50	4	nM	IC50	Affinity Phenotypic Cellular Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Affinity Phenotypic Cellular interaction (Cell Proliferation (Cell TiterGlo assay Promega in NMC 11060 cells)) EUB0000330a BRD2	F	8.4	pIC50	4	nM	IC50	Affinity Phenotypic Cellular Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Inhibition of C-terminal His6-tagged BRD2 BD2 (unknown origin) using H-YSGRGK(Ac)GGK(Ac)GLGK(Ac)-GGAK(Ac)RHRK-Biotin-OH as substrate incubated for 1 hrs by HTRF assay	B	8.41	pIC50	3.93	nM	IC50	J Med Chem (2022) 65: 5760-5799 [PMID:35333526]
bromodomain containing 3/Bromodomain-containing protein 3 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL1795186] [GtoPdb: 2725] [UniProtKB: Q15059]
ChEMBL	Inhibition of human 6x-His-tagged BRD3 bromodomain 2 expressed in Escherichia coli	B	7.09	pKd	82	nM	Kd	J Med Chem (2018) 61: 9316-9334 [PMID:30253095]
ChEMBL	Binding affinity to BRD3-BD2 by isothermal titration calorimetry	B	7.09	pKd	82	nM	Kd	Bioorg Med Chem (2012) 20: 1887-1892 [PMID:22316554]
ChEMBL	Affinity Biochemical interaction (Isothermal titration calorimetry (ITC)) EUB0000330a BRD3	B	7.09	pKd	82	nM	Kd	Affinity Biochemical Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Binding affinity to recombinant BRD3 BD1 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.09	pKd	81	nM	Kd	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Binding affinity to recombinant BRD3 BD2 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.16	pKd	69	nM	Kd	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Binding affinity to BRD3-BD1 by isothermal titration calorimetry	B	7.23	pKd	59.5	nM	Kd	Bioorg Med Chem (2012) 20: 1887-1892 [PMID:22316554]
ChEMBL	Affinity Biochemical interaction (Isothermal titration calorimetry (ITC)) EUB0000330a BRD3	B	7.23	pKd	59.5	nM	Kd	Affinity Biochemical Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Binding affinity of BRD3 (unknown origin) assessed as dissociation constant	B	7.23	pKd	~59	nM	Kd	Nat Commun (2017) 8: 1467-1467 [PMID:29133788]
ChEMBL	Inhibition of human 6x-His-tagged BRD3 bromodomain 1 expressed in Escherichia coli	B	7.23	pKd	59	nM	Kd	J Med Chem (2018) 61: 9316-9334 [PMID:30253095]
ChEMBL	Binding affinity to biotinylated BRD3 BD1 (24 to 144 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells by bio-layer interferometry method	B	7.87	pKd	13.6	nM	Kd	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Binding affinity to biotinylated BRD3 BD2 (306 to 417 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells by bio-layer interferometry method	B	7.93	pKd	11.7	nM	Kd	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Displacement of FAM-labeled ZBA248 from BRD3 BD2 (306 to 417 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells after 30 mins by fluorescence polarization assay	B	8.05	pKi	8.9	nM	Ki	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD3 bromodomain 2 (306 to 417residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	8.05	pKi	8.9	nM	Ki	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Displacement of FAM-labeled ZBA248 from BRD3 BD1 (24 to 144 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells after 30 mins by fluorescence polarization assay	B	8.18	pKi	6.6	nM	Ki	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD3 bromodomain 1 (24 to 144 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	8.18	pKi	6.6	nM	Ki	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Inhibition of recombinant BRD3 BD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.49	pIC50	324	nM	IC50	J Med Chem (2023) 66: 1239-1253 [PMID:36622852]
ChEMBL	Inhibition of BRD3 BD1/2 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.57	pIC50	268	nM	IC50	Eur J Med Chem (2024) 271: 116444-116444 [PMID:38691889]
ChEMBL	Inhibition of BRD3 BD1 (unknown origin) by Alphascreen assay	B	6.64	pIC50	230	nM	IC50	J Med Chem (2024) 67: 18606-18628 [PMID:39356741]
ChEMBL	Inhibition of BRD3 BD2 (unknown origin) by Alphascreen assay	B	6.68	pIC50	210	nM	IC50	J Med Chem (2024) 67: 18606-18628 [PMID:39356741]
ChEMBL	Inhibition of recombinant BRD3 BD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.89	pIC50	129	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
ChEMBL	Inhibition of BRD3 D2 (unknown origin) by competitive fluorescence anisotropy assay	B	7.02	pIC50	96	nM	IC50	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Inhibition of BRD3 BD1 (unknown origin) after 1 hr by TR-FRET assay	B	7.09	pIC50	81	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	7.1	pIC50	80	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	Inhibition of BRD3 D1 (unknown origin) by competitive fluorescence anisotropy assay	B	7.1	pIC50	<80	nM	IC50	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Binding affinity BRD3 BD1 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.1	pIC50	79	nM	IC50	J Med Chem (2022) 65: 2388-2408 [PMID:34982556]
ChEMBL	Inhibition of BRD3 BD1 (unknown origin) incubated for 2 hrs by TR-FRET assay	B	7.12	pIC50	76	nM	IC50	J Med Chem (2021) 64: 18025-18053 [PMID:34908415]
ChEMBL	Binding affinity BRD3 BD2 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.14	pIC50	72	nM	IC50	J Med Chem (2022) 65: 2388-2408 [PMID:34982556]
ChEMBL	Binding affinity to human partial length BRD3 BD1 (P24 to E144 residues) expressed in bacterial expression system using H4K5acIC8acK12acK16ac-biotinylated peptide as substrate preincubated for 30 mins followed by substrate addition by AlphaScreen assay	B	7.16	pIC50	69.2	nM	IC50	J Med Chem (2023) 66: 913-933 [PMID:36577036]
ChEMBL	Inhibition of BRD3 BD2 (unknown origin) after 1 hr by TR-FRET assay	B	7.16	pIC50	69	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Inhibition of BRD3 bromodomain 1 (unknown origin) by Alphascreen based method	B	7.19	pIC50	64	nM	IC50	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Inhibition of human recombinant His-tagged BRD3 BD1 domain expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells by AlphaScreen assay	B	7.19	pIC50	64	nM	IC50	J Med Chem (2020) 63: 3678-3700 [PMID:32153186]
ChEMBL	Inhibition of BRD3 BD2 (unknown origin) incubated for 2 hrs by TR-FRET assay	B	7.21	pIC50	61	nM	IC50	J Med Chem (2021) 64: 18025-18053 [PMID:34908415]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD3 bromodomain 2 (306 to 417residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.25	pIC50	56.7	nM	IC50	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Inhibition of recombinant BRD3 BD2 (unknown origin) incubated for 120 mins by TR-FRET assay	B	7.28	pIC50	53	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD3 bromodomain 1 (24 to 144 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.41	pIC50	39.1	nM	IC50	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Inhibition of human recombinant His-tagged BRD3 BD2 domain expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells by AlphaScreen assay	B	7.41	pIC50	39	nM	IC50	J Med Chem (2020) 63: 3678-3700 [PMID:32153186]
ChEMBL	Inhibition of BRD3 bromodomain 2 (unknown origin) by Alphascreen based method	B	7.41	pIC50	39	nM	IC50	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Inhibition of BRD3-BD1/2 (unknown origin) by HTRF analysis	B	7.42	pIC50	38	nM	IC50	J Med Chem (2023) 66: 2646-2662 [PMID:36774555]
ChEMBL	Inhibition of BRD3 (unknown origin) by TR-FRET assay	B	7.64	pIC50	23	nM	IC50	Medchemcomm (2019) 10: 974-984 [PMID:31303996]
ChEMBL	Binding affinity to human partial length BRD3 BD2 (G306 to P416 residues) expressed in bacterial expression system using H4K5acIC8acK12acK16ac-biotinylated peptide as substrate preincubated for 30 mins followed by substrate addition by AlphaScreen assay	B	8.12	pIC50	7.57	nM	IC50	J Med Chem (2023) 66: 913-933 [PMID:36577036]
ChEMBL	Inhibition of C-terminal His6-tagged BRD3 BD2 (unknown origin) using H-YSGRGK(Ac)GGK(Ac)GLGK(Ac)-GGAK(Ac)RHRK-Biotin-OH as substrate incubated for 1 hrs by HTRF assay	B	8.31	pIC50	4.87	nM	IC50	J Med Chem (2022) 65: 5760-5799 [PMID:35333526]
ChEMBL	Affinity Phenotypic Cellular interaction (Cell Proliferation (Cell TiterGlo assay Promega in NMC 11060 cells)) EUB0000330a BRD3	F	8.4	pIC50	4	nM	IC50	Affinity Phenotypic Cellular Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Affinity Phenotypic Cellular interaction (Cell Proliferation (Cell TiterGlo assay Promega in NMC 11060 cells)) EUB0000330a BRD3	F	8.4	pIC50	4	nM	IC50	Affinity Phenotypic Cellular Literature for EUbOPEN Chemogenomics Library wave 3
bromodomain containing 4/Bromodomain-containing protein 4 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL1163125] [GtoPdb: 1945] [UniProtKB: O60885]
ChEMBL	BROMOscan Binding Assay: A BROMOscan binding assay was utilized to test the in vitro binding activity of (S)-Compounds 1, 2, 3, 4, 5 and 7 to the first and second bromodomains (BRD4(1) and BRD4(2)), separately, of Brd4. (S)-JQ (S8) was used as a positive control.T7 phage strains displaying bromodomains were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection=0.4) and incubated with shaking at 32° C. until lysis (90-150 minutes). The lysates were centrifuged (5,000×g) and filtered (0.2 μm) to remove cell debris. Streptavidin-coated magnetic beads were treated with biotinylated small molecule or acetylated peptide ligands for 30 minutes at room temperature to generate affinity resins for bromodomain assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding.Binding reactions were assembled by combining bromodomains, liganded affinity beads, and test compounds in 1× binding buffer (17% SeaBlock, 0.33×PBS, 0.04% Tween 20, 0.02% BSA, 0.004% Sodium azide, 7.4 mM DTT). Test compounds were prepared as 1000× stocks in 100% DMSO and subsequently serially diluted 1:10 in monoethylene glycol (MEG) to create stocks at 100× the screening concentration (resulting stock solution is 10% DMSO/90% MEG). The compounds were then diluted directly into the assays such that the final concentration of DMSO and MEG were 0.1% and 0.9%, respectively. All reactions were performed in polystyrene 96-well plates in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM nonbiotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The bromodomain concentration in the eluates was measured by qPCR.	B	5.44	pKd	3600	nM	Kd	US-10124009-B2. Bromodomain inhibitors (2018)
ChEMBL	Binding affinity to BRD4 BD2 (unknown origin) assessed as dissociation constant by ITC analysis	B	6.64	pKd	230	nM	Kd	Eur J Med Chem (2021) 222: 113588-113588 [PMID:34107385]
ChEMBL	Binding affinity to BRD4 C-terminal bromodomain 2 H437D mutant (unknown origin) expressed in Escherichia coli Bl21(DE3) by fluorescence polarization assay	B	6.7	pKd	200	nM	Kd	J Med Chem (2018) 61: 9316-9334 [PMID:30253095]
ChEMBL	Binding affinity to BRD4 D2 (unknown origin) assessed as dissociation constant by isothermal titration calorimetry	B	6.89	pKd	130	nM	Kd	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Binding affinity to BRD4 C-terminal bromodomain 2 (unknown origin) expressed in Escherichia coli Bl21(DE3)	B	6.92	pKd	120	nM	Kd	J Med Chem (2018) 61: 9316-9334 [PMID:30253095]
ChEMBL	Binding affinity to his-tagged human BRD4 BD2 assessed as dissociation constant by SPR analysis	B	6.92	pKd	119	nM	Kd	J Med Chem (2024) 67: 2712-2731 [PMID:38295759]
ChEMBL	Binding affinity to human BRD4 expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells by isothermal titration calorimetry-based assay	B	7	pKd	100	nM	Kd	J Med Chem (2018) 61: 504-513 [PMID:28595007]
ChEMBL	Binding affinity to BRD4 BD2 (unknown origin) by ITC analysis	B	7.02	pKd	96.1	nM	Kd	J Med Chem (2024) 67: 2712-2731 [PMID:38295759]
ChEMBL	Binding affinity to his6-tagged human BRD4 BD1 (44 to 168 residues) expressed in Escherichia coli Rossetta BL21 (DE3) cells assessed as dissociation constant by SPR analysis	B	7.02	pKd	95.2	nM	Kd	J Med Chem (2024) 67: 2712-2731 [PMID:38295759]
ChEMBL	Binding affinity to recombinant BRD4 BD1 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.04	pKd	92	nM	Kd	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Binding affinity to human His6-tagged BRD4 BD1 (N44 to E168 residues) expressed in Escherichia coli BL21 (DE3) cells by isothermal titration calorimetry method	B	7.04	pKd	92	nM	Kd	ACS Med Chem Lett (2018) 9: 262-267 [PMID:29541371]
ChEMBL	Binding affinity to BRD4-BD2 by isothermal titration calorimetry	B	7.05	pKd	90.1	nM	Kd	Bioorg Med Chem (2012) 20: 1887-1892 [PMID:22316554]
ChEMBL	Affinity Biochemical interaction (Isothermal titration calorimetry (ITC)) EUB0000330a BRD4	B	7.05	pKd	90.1	nM	Kd	Affinity Biochemical Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Inhibition of human 6x-His-tagged BRD4 bromodomain 2 expressed in Escherichia coli	B	7.05	pKd	90	nM	Kd	J Med Chem (2018) 61: 9316-9334 [PMID:30253095]
ChEMBL	Binding affinity to BRD4 BD2 domain (unknown origin) by isothermal titration calorimetry assay	B	7.05	pKd	90	nM	Kd	Eur J Med Chem (2020) 207: 112750-112750 [PMID:32871345]
ChEMBL	Binding affinity to recombinant human N-terminal hexaHis-tagged BRD4 expressed in Escherichia coli BL21 (DE3) cells by ITC analysis	B	7.15	pKd	71	nM	Kd	J Med Chem (2021) 64: 15772-15786 [PMID:34710325]
ChEMBL	Binding affinity to recombinant BRD4 BD2 (unknown origin) incubated for 1 hr by TR-FRET assay	B	7.21	pKd	62	nM	Kd	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Binding affinity to BRD4 by isothermal titration calorimetry	B	7.22	pKd	60	nM	Kd	Bioorg Med Chem (2012) 20: 1878-1886 [PMID:22137933]
ChEMBL	Binding affinity to BRD4 BD1 (unknown origin) assessed as dissociation constant by ITC analysis	B	7.29	pKd	51	nM	Kd	Eur J Med Chem (2021) 222: 113588-113588 [PMID:34107385]
GtoPdb	Displacement binding constant to recombinant BRD4-BD1 protein.	-	7.3	pKd	50	nM	Kd	Nature (2010) 468: 1067-73 [PMID:20871596]
ChEMBL	Binding affinity to BRD4 bromodomain 1 (unknown origin) by isothermal titration calorimetry	B	7.3	pKd	50	nM	Kd	Medchemcomm (2018) 9: 1779-1802 [PMID:30542529]
ChEMBL	Binding affinity to BRD4-BD1 by isothermal titration calorimetry	B	7.31	pKd	49	nM	Kd	Bioorg Med Chem (2012) 20: 1887-1892 [PMID:22316554]
ChEMBL	Binding affinity to BRD4-BD1 by isothermal titration calorimetry	B	7.31	pKd	49	nM	Kd	Bioorg Med Chem (2012) 20: 1878-1886 [PMID:22137933]
ChEMBL	Binding affinity to BRD4 isoform 1	B	7.31	pKd	49	nM	Kd	J Med Chem (2012) 55: 9393-9413 [PMID:22924434]
ChEMBL	Inhibition of human 6x-His-tagged BRD4 bromodomain 1 expressed in Escherichia coli	B	7.31	pKd	49	nM	Kd	J Med Chem (2018) 61: 9316-9334 [PMID:30253095]
ChEMBL	Binding affinity to BRD4 (unknown origin)	B	7.31	pKd	49	nM	Kd	Bioorg Med Chem Lett (2020) 30: 126958-126958 [PMID:32019712]
ChEMBL	Affinity Biochemical interaction (Isothermal titration calorimetry (ITC)) EUB0000330a BRD4	B	7.31	pKd	49	nM	Kd	Affinity Biochemical Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Binding affinity to BRD4 BD1 domain (unknown origin) by isothermal titration calorimetry assay	B	7.31	pKd	49	nM	Kd	Eur J Med Chem (2020) 207: 112750-112750 [PMID:32871345]
ChEMBL	Binding affinity to BRD4 BD1 (unknown origin) by ITC analysis	B	7.31	pKd	49	nM	Kd	J Med Chem (2024) 67: 2712-2731 [PMID:38295759]
ChEMBL	Binding affinity of BRD4 (unknown origin) assessed as dissociation constant	B	7.31	pKd	~49	nM	Kd	Nat Commun (2017) 8: 1467-1467 [PMID:29133788]
ChEMBL	Binding affinity to recombinant human N-terminal hexaHis-tagged BRD4 expressed in Escherichia coli BL21 (DE3) cells by MST assay	B	7.46	pKd	35	nM	Kd	J Med Chem (2021) 64: 15772-15786 [PMID:34710325]
ChEMBL	Binding affinity to human His6-tagged BRD4 bromodomain 1 (N44 to E168 residues) expressed in Escherichia coli BL21(DE3) cells by isothermal titration calorimetry	B	7.47	pKd	34	nM	Kd	J Med Chem (2016) 59: 1565-1579 [PMID:26731490]
ChEMBL	Binding affinity towards BRD4-BD1 (unknown origin) measured by isothermal titration calorimetry (ITC) method	B	7.57	pKd	27.2	nM	Kd	ACS Med Chem Lett (2022) 13: 1621-1627 [PMID:36262390]
ChEMBL	Binding affinity to BRD4 D1 (unknown origin) assessed as dissociation constant by isothermal titration calorimetry	B	7.72	pKd	19	nM	Kd	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Binding affinity to BRD4 BD2 (unknown origin) by thermal denaturation assay	B	7.85	pKd	14	nM	Kd	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Binding affinity to human BRD4 BD1 (N44 to E168 aa residues) by bromoKdELECT Discover assay	B	7.89	pKd	13	nM	Kd	J Med Chem (2020) 63: 15603-15620 [PMID:33275431]
ChEMBL	Binding affinity to biotinylated BRD4 BD1 (44 to 168 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells by bio-layer interferometry method	B	7.89	pKd	12.8	nM	Kd	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Binding affinity to BRD4 BD1 (unknown origin) by thermal denaturation assay	B	8.05	pKd	9	nM	Kd	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Binding affinity to recombinant human N-terminal hexaHis-tagged BRD4 expressed in Escherichia coli BL21 (DE3) cells by qPCR assay	B	8.09	pKd	8.2	nM	Kd	J Med Chem (2021) 64: 15772-15786 [PMID:34710325]
ChEMBL	Binding affinity to biotinylated BRD4 BD2 (333 to 460 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells by bio-layer interferometry method	B	8.17	pKd	6.7	nM	Kd	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Binding affinity to human N-terminal His6-tagged BRD4 BD1-BD2 (K57 to K550 residues) after 1 hr using alexa-647 conjugated probe by TR-FRET assay	B	7.11	pKi	77	nM	Ki	J Med Chem (2017) 60: 8369-8384 [PMID:28949521]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD4 bromodomain 1 (44 to 168 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.83	pKi	14.9	nM	Ki	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant human N-terminal His6-tagged BRD4 bromodomain 1 (44 to 168 residues) expressed in Rosetta2 DE3 cells after 30 mins by Flourescence polarization assay	B	7.83	pKi	14.9	nM	Ki	J Med Chem (2017) 60: 3887-3901 [PMID:28463487]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant human N-terminal His6-tagged BRD4 bromodomain 2 (333 to 460 residues) expressed in Rosetta2 DE3 cells after 30 mins by Flourescence polarization assay	B	7.92	pKi	12	nM	Ki	J Med Chem (2017) 60: 3887-3901 [PMID:28463487]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD4 bromodomain 2 (333 to 460 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.92	pKi	12	nM	Ki	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Displacement of FAM-labeled ZBA248 from BRD4 BD2 (333 to 460 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells after 30 mins by fluorescence polarization assay	B	7.97	pKi	10.7	nM	Ki	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Displacement of FAM-labeled ZBA248 from BRD4 BD1 (44 to 168 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells after 30 mins by fluorescence polarization assay	B	8.12	pKi	7.6	nM	Ki	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Binding affinity to human BRD4 BD1 (44 to 168 residues) by fluorescence polarization assay	B	8.28	pKi	5.3	nM	Ki	J Med Chem (2018) 61: 6685-6704 [PMID:30019901]
ChEMBL	Inhibition of BRD4 (unknown origin) by biotinylated-JQ1 based AlphaScreen BRD binding assay	B	4.09	pIC50	81500	nM	IC50	J Med Chem (2014) 57: 9019-9027 [PMID:25314271]
ChEMBL	Inhibition of BRD4 in human SAE cells assessed as reduction in TLR3 agonist poly(I:C) -induced ISG56 RNA expression preincubated for 24 hrs followed by poly(I:C) addition and measured after 4 hrs by SYBR green dye-based qRT-PCR analysis	B	5.79	pIC50	1630	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Inhibition of BRD4 in human SAE cells assessed as reduction in TLR3 agonist poly(I:C) -induced IL-8 RNA expression preincubated for 24 hrs followed by poly(I:C) addition and measured after 4 hrs by SYBR green dye-based qRT-PCR analysis	B	5.82	pIC50	1510	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Inhibition of BRD4 in human SAE cells assessed as reduction in TLR3 agonist poly(I:C) -induced grobeta RNA expression preincubated for 24 hrs followed by poly(I:C) addition and measured after 4 hrs by SYBR green dye-based qRT-PCR analysis	B	5.83	pIC50	1490	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Inhibition of BRD4 in human 90-8TL cells assessed as reduction in cell viability at 4.9 to 2500 nM incubated for 72 hrs by SRB assay	B	5.85	pIC50	1422	nM	IC50	PLoS One (2017) 12: 183155-183155 [PMID:28813519]
ChEMBL	Inhibition of BRD4 in human SAE cells assessed as reduction in TLR3 agonist poly(I:C) -induced ISG54 RNA expression preincubated for 24 hrs followed by poly(I:C) addition and measured after 4 hrs by SYBR green dye-based qRT-PCR analysis	B	5.86	pIC50	1380	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Inhibition of BRD4 in human T265 cells assessed as reduction in cell viability at 4.9 to 2500 nM incubated for 72 hrs by SRB assay	B	5.87	pIC50	1364.2	nM	IC50	PLoS One (2017) 12: 183155-183155 [PMID:28813519]
ChEMBL	Inhibition of BRD4 in human SNF96.2 cells assessed as reduction in cell viability at 4.9 to 2500 nM incubated for 72 hrs by SRB assay	B	5.92	pIC50	1215.5	nM	IC50	PLoS One (2017) 12: 183155-183155 [PMID:28813519]
ChEMBL	Inhibition of BRD4 in poly(I:C)-stimulated human SAECs TLR3-inducible IL-6 gene expression pre-incubated for 24 hrs before poly(I:C) stimulation for 4 hrs by qRT-PCR analysis	B	5.99	pIC50	1020	nM	IC50	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Inhibition of BRD4 in poly(I:C)-stimulated human SAECs TLR3-inducible CIG5 gene expression pre-incubated for 24 hrs before poly(I:C) stimulation for 4 hrs by qRT-PCR analysis	B	6.02	pIC50	950	nM	IC50	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Inhibition of recombinant human N-terminal His-tagged BRD4 bromodomain 1 (49 to 170 residues) expressed in Escherichia coli using biotinylated peptide containing acetylated lysine as substrate pretreated for 15 mins followed by substrate addition after 1 hr by TR-FRET assay	B	6.09	pIC50	820	nM	IC50	Bioorg Med Chem (2017) 25: 3677-3684 [PMID:28549889]
ChEMBL	Displacement of Fl-JQ1 from BRD4 C-terminal bromodomain 2 H437D mutant (unknown origin) expressed in Escherichia coli Bl21(DE3) by fluorescence polarization assay	B	6.23	pIC50	590	nM	IC50	J Med Chem (2018) 61: 9316-9334 [PMID:30253095]
ChEMBL	In Vitro Enzyme Inhibition Assay-BRD4 Inhibition: Inhibition of BRD4 was determined as previously described in U.S. Pat. No. 9,034,900.	B	6.3	pIC50	<500	nM	IC50	US-10202360-B2. Therapeutic compounds (2019)
ChEMBL	In Vitro Enzyme Inhibition Assay-BRD4 Inhibition: Inhibition of BRD4 was determined as described previously. See, e.g., U.S. Pat. No. 9,034,900.	B	6.3	pIC50	<500	nM	IC50	US-10617680-B2. Therapeutic compounds (2020)
ChEMBL	In Vitro Enzyme Inhibition Assay-BRD4 Inhibition: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BRD4 was cloned, expressed and purified to homogeneity (P. Filipakopoulos et al. Nature 468, 1067-1073, 2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 minute incubation at room temperature, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After two hours of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.	B	6.3	pIC50	<500	nM	IC50	US-11020380-B2. Therapeutic compounds (2021)
ChEMBL	Inhibition of BRD4 bromodomain 2 (unknown origin) expressed in Escherichia coli Bl21(DE3) by fluorescence anisotropy	B	6.31	pIC50	490	nM	IC50	J Med Chem (2018) 61: 9316-9334 [PMID:30253095]
ChEMBL	Inhibition of BRD4-BD1 (unknown origin) using C-terminal biotinylated tetra-acetylated histone H4 peptide as substrate incubated for 2.5 hrs by alphascreen assay	B	6.43	pIC50	370	nM	IC50	Bioorg Med Chem (2021) 55: 116592-116592 [PMID:34999525]
ChEMBL	Inhibition of fluorescent-tagged ligand from nanoLuc fused full length BRD4 (unknown origin) expressed in 293T cells by luciferase reporter gene based BRET engagement assay	B	6.52	pIC50	300	nM	IC50	J Med Chem (2018) 61: 9301-9315 [PMID:30289257]
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	6.65	pIC50	224	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	Displacement of APC-labeled biotinylated-avidin from Euphorium-chelated recombinant human BRD4 bromodomain 1 expressed in Escherichia coli preincubated for 15 mins followed by APC-labeled biotinylated-avidin addition and measured after 1 hr under dark condition by TR-FRET assay	B	6.7	pIC50	199	nM	IC50	J Med Chem (2020) 63: 7186-7210 [PMID:32453591]
ChEMBL	Inhibition of biotinylated H4 K5,8,12,16Ac peptide binding to GST-tagged BRD4 bromodomain1 (unknown origin) measured after 60 mins by AlphaScreen assay	B	6.77	pIC50	170	nM	IC50	Bioorg Med Chem (2019) 27: 1871-1881 [PMID:30926312]
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	6.77	pIC50	170	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	Inhibition of BRD4 (unknown origin) by TR-FRET assay	B	6.84	pIC50	144	nM	IC50	Medchemcomm (2019) 10: 974-984 [PMID:31303996]
ChEMBL	Binding affinity to BRD4 BD1 (unknown origin) incubated for 1 hr in presence of fluorescein labeled tracer by fluorescence polarization assay	B	6.89	pIC50	129	nM	IC50	J Med Chem (2024) 67: 14125-14154 [PMID:39132814]
ChEMBL	Inhibition of N-terminal his6-tagged human BRD4 BD2 incubated for 15 mins by TR-FRET assay	B	6.9	pIC50	126.2	nM	IC50	J Med Chem (2024) 67: 2712-2731 [PMID:38295759]
ChEMBL	Displacement of APC-labeled biotinylated-avidin from Euphorium-chelated recombinant human BRD4 bromodomain 2 expressed in Escherichia coli preincubated for 15 mins followed by APC-labeled biotinylated-avidin addition and measured after 1 hr under dark condition by TR-FRET assay	B	6.9	pIC50	126	nM	IC50	J Med Chem (2020) 63: 7186-7210 [PMID:32453591]
ChEMBL	Displacement of FITC-conjugated JQ1 from BRD4 BD2 (unknown origin) expressed in Escherichia coli BL21-DE3 rosetta cells incubated for 15 mins by competitive fluorescence polarization assay	B	6.91	pIC50	123	nM	IC50	J Med Chem (2021) 64: 11637-11650 [PMID:34279939]
ChEMBL	Inhibition of His6-tagged human BRD4 bromodomain-1 (N44 to E168 residues) expressed in Escherichia coli BL21(DE3) using biotinylated-H4KAc4 as substrate by AlphaScreen assay	B	6.92	pIC50	120	nM	IC50	Eur J Med Chem (2018) 152: 542-559 [PMID:29758518]
ChEMBL	Inhibition of human His6-tagged BRD4 BD1 (N44 to E168 residues) expressed in Escherichia coli BL21 (DE3) cells using H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK-Biotin-OH as substrate after 2.5 hrs by AlphaScreen assay	B	6.92	pIC50	120	nM	IC50	ACS Med Chem Lett (2018) 9: 262-267 [PMID:29541371]
ChEMBL	Inhibition of human His6-tagged BRD4 bromodomain 1 (N44 to E168 residues) expressed in Escherichia coli BL21(DE3) cells using biotin-H4KAc4 as substrate incubated for 2 hrs by alphascreen assay	B	6.92	pIC50	120	nM	IC50	J Med Chem (2016) 59: 1565-1579 [PMID:26731490]
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	6.94	pIC50	114	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	Inhibition of BRD4 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.95	pIC50	111	nM	IC50	Eur J Med Chem (2024) 271: 116444-116444 [PMID:38691889]
ChEMBL	Inhibition of BRD4 BD1/2 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.96	pIC50	109	nM	IC50	Eur J Med Chem (2024) 271: 116444-116444 [PMID:38691889]
ChEMBL	Inhibitory Activity Assay of BRD4 Small Molecule Compounds: AlphaScreen kit (Perkin Elmer) was used to detect effect of the compounds on binding of BRD4 bromodomain to acetylated histone H4 polypeptide. The bromodomain BRD4_BD1 (49-170) protein constructed by recombination (Org. Biomol. Chem., 2017, 15, 9352-9361) has a purity greater than 95%, and contains hexahistine tag, namely (His)6tag (hereinafter referred to as (His)6tag), at the N-terminus of the amino acid sequence thereof. Each of fusion proteins containing (His)6tag can be recognized and bound by Ni2+. The acetylated histone H4 polypeptide was provided by Suzhou Qiangyao Biological Technology Co., Ltd, and the sequence was N-C: SGRG-K(Ac)-GG-K(Ac)-GLG-K(Ac)-GGA-K(Ac) RHRKVGG-K (biotin), in which the lysine at positions 5, 8, 12 and 16 was acetylated, and the C-terminus of polypeptide was marked with biotin 5-(2-oxohexahydro-H-thieno[3,4-d]imidazole-4-yl)pentanoic acid, and the purity is greater than 95%. Donor microbeads and acceptor microbeads were purchased from Perkin Elmer (AlphaScreen Histidine Detection Kit (Nickle Chelate) 6760619M Lot: 2236078). The donor microbeads were coated with streptavidin and can bind to biotinylated acetylated H4 polypeptide. The acceptor microbeads were coated with Ni2+ ions and can bind to the BD1 protein with (His)6tag. When the BD1 protein recognized the acetylated H4 polypeptide, the donor microbeads and the acceptor microbeads can be brought closer to a certain distance. Within this range, the donor microbeads can produce singlet oxygen after being irradiated with a 680 nm excitation light, and transfer the singlet oxygen to the acceptor microbeads. After a series of cascade chemical reactions, the acceptor microbeads will generate an emission wave of 520-620 nm, which in turn can be detected a signal. In this experiment, the test system was 20 μL, and the compound needs to be diluted in buffer 1 (20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM dithiothreitol). First, a solution of 20 mM compound was consecutively diluted twice, each in 10 times, totally in 100 times to a concentration of 200 μM. Then the solution of 200 μM compound was serially diluted in three times in the buffer 1 containing 1/100 DMSO to obtain a 8× working solution of a compound dilution series with a compound concentration of 200 μM to 10 nM (final concentrations: 25.0 μM, 8.33 μM, 2.77 μM, 0.926 μM, 0.309 μM, 0.103 μM, 0.0343 μM, 0.0114 μM, 0.00381 μM, 0.00127 μM). The positive compound used in the test was JQ (Nature 2010, 468, 1067-1073), which was purchased from Sigma. 2.5 μL of the compound solution was added to a white 384-well plate (OptiPlate-384, PerkinElmer 6007299). The recombinant BD1 protein solution and the acetylated histone H4 polypeptide were diluted in the buffer 2 (20 mM HEPES pH 7.4, 150 mM NaCl, 0.01% Triton X-100, 0.1% bovine serum protein (w/v, Sigma), 1 mM dithiothreitol) to 100 nM and 100 nM respectively, to obtain a 8×BD1 protein working solution and a 4× acetylated histone H4 polypeptide working solution. The donor microbeads and the acceptor microbeads are diluted together in the buffer 2, both in the ratio of 1:100, to obtain a 2× microbead mixed working solution. The plate was added with 2.5 μL of BD1 protein working solution and incubated with the compound for 20 min at room temperature, and then added with 5 μL of acetylated histone H4 polypeptide working solution and incubated at room temperature for 5 min, and finally added with 10 μL of microbead mixed working solution and incubated at room temperature for 60 min. Then the signals were read on an EnVision microplate reader (Perkin Elmer) (excitation wavelength was 680 nM, and detection wavelength was 520-620 nM). IC50 values of the compounds at different concentrations for inhibiting the binding of BD1 protein to acetylated H4 polypeptide were calculated by fitting using GraphPad Prism 5.0 software.	B	6.99	pIC50	103	nM	IC50	US-11427593-B2. Bromodomain inhibitor compound and use thereof (2022)
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	6.99	pIC50	103	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	7.02	pIC50	96	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	Inhibition of BRD4 D2 (unknown origin) by competitive fluorescence anisotropy assay	B	7.03	pIC50	94	nM	IC50	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) incubated for 120 mins by TR-FRET assay	B	7.03	pIC50	94	nM	IC50	Eur J Med Chem (2024) 271: 116444-116444 [PMID:38691889]
ChEMBL	Inhibition of BRD4 D1 (unknown origin) by competitive fluorescence anisotropy assay	B	7.04	pIC50	<92	nM	IC50	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) after 1 hr by TR-FRET assay	B	7.04	pIC50	92	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Binding affinity recombinant human BRD4 BD1 (42 to 168 residues) expressed in Escherichia coli BL21(DE3) incubated for 1 hr by TR-FRET assay	B	7.04	pIC50	92	nM	IC50	J Med Chem (2022) 65: 2388-2408 [PMID:34982556]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) incubated for 2 hrs by TR-FRET assay	B	7.04	pIC50	92	nM	IC50	J Med Chem (2021) 64: 18025-18053 [PMID:34908415]
ChEMBL	Inhibition of human His-tagged BRD4 BD1 (49 to 170 residues) using H-SGRGK(Ac)GGK(Ac)GLGK-(Ac)GGAK(Ac)RHRK(Biotin)-OH as substrate preincubated for 30 mins followed by substrate addition measured after 30 mins by TR-FRET assay	B	7.04	pIC50	91	nM	IC50	Eur J Med Chem (2016) 121: 294-299 [PMID:27266999]
ChEMBL	Inhibition of human BRD4 expressed in Escherichia coli BL21(DE3)-R3-pRARE2 by bromodomain alphascreen peptide displacement assay	B	7.05	pIC50	90	nM	IC50	Bioorg Med Chem Lett (2016) 26: 2931-2935 [PMID:27142751]
ChEMBL	Inhibition of BRD4 bromodomain 1/2 (unknown origin) by alphascreen assay	B	7.05	pIC50	90	nM	IC50	Bioorg Med Chem Lett (2019) 29: 126577-126577 [PMID:31421967]
ChEMBL	Inhibition of recombinant BRD4 BD2 (unknown origin) incubated for 120 mins by TR-FRET assay	B	7.05	pIC50	89	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	7.05	pIC50	89	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	Inhibition of BRD4-BD1 (44 to 168 residues) (unknown origin) using histone H4 peptide as substrate preincubated for 15 mins followed by addition of substrate measured after 60 mins by AlphaScreen assay	B	7.06	pIC50	88	nM	IC50	Bioorg Med Chem (2017) 25: 2482-2490 [PMID:28314513]
ChEMBL	Inhibition of BRD4 bromodomain1 (unknown origin) using peptide H4 as substrate incubated for 15 mins followed by substrate addition and measured after 1 hr by alphascreen assay	B	7.06	pIC50	87	nM	IC50	ACS Med Chem Lett (2019) 10: 1680-1685 [PMID:31857846]
ChEMBL	Inhibition of BRD4-BD2 (333 to 460 residues) (unknown origin) using histone H4 peptide as substrate preincubated for 15 mins followed by addition of substrate measured after 60 mins by AlphaScreen assay	B	7.06	pIC50	87	nM	IC50	Bioorg Med Chem (2017) 25: 2482-2490 [PMID:28314513]
ChEMBL	Inhibition of BRD4 in human HL60 cells assessed as reduction in C-myc production after 24 hrs by ELISA	B	7.07	pIC50	86	nM	IC50	Bioorg Med Chem (2017) 25: 3677-3684 [PMID:28549889]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) incubated for 15 mins by TR-FRET method	B	7.07	pIC50	86	nM	IC50	Bioorg Med Chem (2023) 91: 117386-117386 [PMID:37379621]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) by TR-FRET assay	B	7.07	pIC50	85	nM	IC50	Bioorg Med Chem Lett (2024) 109: 129848-129848 [PMID:38876176]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin)	B	7.07	pIC50	85	nM	IC50	Bioorg Med Chem Lett (2024) 109: 129849-129849 [PMID:38876177]
ChEMBL	Inhibition of BRD4 BD1/BD2 (unknown origin) incubated for 2 hrs by TR-FRET assay	B	7.08	pIC50	84	nM	IC50	J Med Chem (2021) 64: 18025-18053 [PMID:34908415]
ChEMBL	Inhibition of BRD4-BD1/2 (unknown origin)	B	7.08	pIC50	83	nM	IC50	Eur J Med Chem (2022) 230: 114116-114116 [PMID:35091172]
ChEMBL	Inhibition of recombinant BRD4 (unknown origin) incubated for 120 mins by TR-FRET assay	B	7.08	pIC50	83	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) by Alphascreen assay	B	7.1	pIC50	80	nM	IC50	J Med Chem (2024) 67: 18606-18628 [PMID:39356741]
ChEMBL	Inhibition of biotinylated H4 K5,8,12,16Ac peptide binding to GST-tagged BRD4 bromodomain2 (unknown origin) measured after 60 mins by AlphaScreen assay	B	7.1	pIC50	80	nM	IC50	Bioorg Med Chem (2019) 27: 1871-1881 [PMID:30926312]
ChEMBL	Inhibition of BRD4 bromodomain2 (unknown origin) using peptide H4 as substrate incubated for 15 mins followed by substrate addition and measured after 1 hr by alphascreen assay	B	7.1	pIC50	79	nM	IC50	ACS Med Chem Lett (2019) 10: 1680-1685 [PMID:31857846]
ChEMBL	Inhibition of BRD4 (unknown origin) by HTRF analysis	B	7.11	pIC50	78	nM	IC50	J Med Chem (2023) 66: 2646-2662 [PMID:36774555]
ChEMBL	Displacement of acetylated histone peptide from BRD4-BD1 by luminescence proximity homogenous assay	B	7.11	pIC50	77	nM	IC50	Bioorg Med Chem (2012) 20: 1887-1892 [PMID:22316554]
ChEMBL	Binding affinity to first bromodomain of BRD4	B	7.11	pIC50	77	nM	IC50	ACS Med Chem Lett (2012) 3: 691-694 [PMID:24900532]
ChEMBL	Displacement of H4(1-21)KAc5,8,12,16 peptide from BRD4 isoform 1 bead based amplified luminescent proximity homogeneous assay	B	7.11	pIC50	77	nM	IC50	J Med Chem (2012) 55: 9393-9413 [PMID:22924434]
ChEMBL	Inhibition of human N-terminal BRD4 bromodomain expressed in Escherichia coli BL21(DE3) after 30 mins by luminescence proximity homogeneous assay	B	7.11	pIC50	77	nM	IC50	J Med Chem (2012) 55: 587-596 [PMID:22136469]
ChEMBL	Binding affinity to human BRD4 bromodomain 1 using H4Ac4 peptide by amplified luminescent proximity homogeneous assay	B	7.11	pIC50	77	nM	IC50	J Med Chem (2013) 56: 3217-3227 [PMID:23517011]
ChEMBL	Inhibition of BRD4 bromodomain 1 (unknown origin)-acetylated histone H4 interaction by luminescence proximity homogeneous assay	B	7.11	pIC50	77	nM	IC50	Medchemcomm (2012) 3: 123-134
ChEMBL	Inhibition of recombinant His-tagged human BRD4 bromodomain 1 expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells preincubated for 30 mins followed by H4K5acK8acK12acK16ac substrate addition measured after 30 mins by AlphaScreen assay	B	7.11	pIC50	77	nM	IC50	Bioorg Med Chem (2018) 26: 2937-2957 [PMID:29776834]
ChEMBL	Inhibition of recombinant human His6-tagged BRD4 bromodomain 1 expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells preincubated for 30 mins followed by H4K5acK8acK12acK16ac peptide substrate addition after 30 mins by alphascreen assay	B	7.11	pIC50	77	nM	IC50	J Med Chem (2017) 60: 4533-4558 [PMID:28195723]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) by alphascreen assay	B	7.11	pIC50	77	nM	IC50	Bioorg Med Chem (2024) 106: 117752-117752 [PMID:38749341]
ChEMBL	Inhibition of human BRD4 BD1 by TR-FRET assay	B	7.11	pIC50	77	nM	IC50	J Med Chem (2024) 67: 922-951 [PMID:38214982]
ChEMBL	Inhibition of BDR4 BD1 (unknown origin)	B	7.11	pIC50	77	nM	IC50	Eur J Med Chem (2024) 263: 115924-115924 [PMID:37992518]
ChEMBL	Inhibition of human His-tagged BRD4 bromodomain 1 expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells using biotin-H4K5acK8acK12acK16ac as substrate preincubated for 30 mins followed by substrate addition measured after 30 mins by alphascreen assay	B	7.11	pIC50	77	nM	IC50	J Med Chem (2016) 59: 1565-1579 [PMID:26731490]
ChEMBL	Inhibition of BRD4(1) (unknown origin)	B	7.11	pIC50	77	nM	IC50	Bioorg Med Chem Lett (2016) 26: 2931-2935 [PMID:27142751]
ChEMBL	Inhibition of BRD4 bromodomain 1 (unknown origin) by alpha-screen method	B	7.11	pIC50	77	nM	IC50	Medchemcomm (2018) 9: 1779-1802 [PMID:30542529]
ChEMBL	Inhibition of HSGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH binding to recombinant human His6-tagged BRD4 bromodomain 1 expressed in Escherichia coli BL21 (DE3)-R3-pRARE2 cells preincubated for 30 mins followed by HSGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH addition and measured after 30 mins by Alphascreen assay	B	7.11	pIC50	77	nM	IC50	J Med Chem (2020) 63: 3956-3975 [PMID:32208600]
ChEMBL	Inhibition of BRD4 (unknown origin)	B	7.11	pIC50	77	nM	IC50	Eur J Med Chem (2020) 186: 111852-111852 [PMID:31759729]
ChEMBL	Inhibition of BRD4 (unknown origin)	B	7.11	pIC50	77	nM	IC50	Bioorg Med Chem Lett (2020) 30: 126958-126958 [PMID:32019712]
ChEMBL	Inhibition of BRD4-BD1 domain (unknown origin) assessed as inhibition of tetra-acetylated Histone H4 peptide binding to BRD4 by ALPHA-screen assay	B	7.11	pIC50	77	nM	IC50	Eur J Med Chem (2020) 207: 112750-112750 [PMID:32871345]
ChEMBL	Selectivity interaction (AlphaScreen) EUB0000330a BRD4	B	7.11	pIC50	77	nM	IC50	Selectivity Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Inhibition of human His-tagged BRD4 BD1 domain using H4K5acK8acK12acK16ac as substrate preincubated for 30 mins followed by substrate addition measured after 30 mins by AlphaScreen assay	B	7.11	pIC50	77	nM	IC50	Eur J Med Chem (2020) 208: 112780-112780 [PMID:32883643]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) by Alpha screen assay	B	7.11	pIC50	77	nM	IC50	Eur J Med Chem (2022) 227: 113953-113953 [PMID:34731760]
ChEMBL	Inhibition of HYSGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH binding to recombinant human His6-tagged BRD4 bromodomain 1 (N44 to E168 residues) expressed in Escherichia coli BL21 (DE3) cells measured after 2 hrs by Alphascreen assay	B	7.11	pIC50	77	nM	IC50	J Med Chem (2018) 61: 3037-3058 [PMID:29566488]
ChEMBL	Inhibition of His tagged human BRD4 BD1 expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells using biotinylated H4K5acK8acK12acK16ac peptide as substrate pretreated for 30 mins followed by substrate addition and measured after 30 mins by Alphascreen analysis	B	7.11	pIC50	77	nM	IC50	Eur J Med Chem (2022) 238: 114423-114423 [PMID:35544982]
ChEMBL	Binding affinity to BRD4 BD1 (unknown origin) by fluorescence anisotropy method	B	7.11	pIC50	77	nM	IC50	J Med Chem (2021) 64: 10497-10511 [PMID:34236185]
ChEMBL	Inhibition of BRD4 bromodomain-1 (unknown origin) by AlphaScreen assay	B	7.11	pIC50	77	nM	IC50	Eur J Med Chem (2018) 152: 542-559 [PMID:29758518]
ChEMBL	Displacement of APC-labeled biotinylated-avidin from Euphorium-chelated recombinant human N-terminal GST-tagged BRD4 bromodomain 1 expressed in Escherichia coli preincubated for 15 mins followed by APC-labeled biotinylated-avidin addition and measured after 1 hr under dark condition by TR-FRET assay	B	7.13	pIC50	74	nM	IC50	Bioorg Med Chem (2020) 28: 115601-115601 [PMID:32631570]
ChEMBL	Binding affinity to BRD4 BD2 (unknown origin) by fluorescence anisotropy method	B	7.14	pIC50	72	nM	IC50	J Med Chem (2021) 64: 10497-10511 [PMID:34236185]
ChEMBL	Inhibition of JQ1-FITC binding to His6-tagged BRD4-BD1 (unknown origin) expressed in Escherichia coli BL21 (DE3)-codon plus-RIL cells incubated in dark for 4 hrs by fluorescence anisotropy assay	B	7.15	pIC50	70	nM	IC50	Eur J Med Chem (2017) 137: 176-195 [PMID:28586718]
ChEMBL	Inhibition of BRD4 bromodomain 2 (unknown origin) by Alphascreen based method	B	7.16	pIC50	69	nM	IC50	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Inhibition of BRD4 in human Raji cells assessed as reduction of MYC expression after 4 hrs	B	7.16	pIC50	69	nM	IC50	ACS Med Chem Lett (2013) 4: 835-840 [PMID:24900758]
ChEMBL	Inhibition of human recombinant His-tagged BRD4 BD2 domain using H-SGRGK(Ac)GGK(Ac)GLGK-(Ac)GGAK(Ac)RHRK(Biotin)-OH as substrate preincubated with enzyme for 30 mins followed by substrate addition measured after 30 mins by AlphaScreen assay	B	7.16	pIC50	69	nM	IC50	J Med Chem (2020) 63: 3678-3700 [PMID:32153186]
ChEMBL	Inhibition of BRD4 (unknown origin) using BET peptide as substrate preincubated for 15 mins followed by substrate addition and measured after 60 mins by HTRF assay	B	7.16	pIC50	68.9	nM	IC50	Bioorg Med Chem (2024) 101: 117609-117609 [PMID:38364599]
ChEMBL	Displacement of fluorescein-labeled JQ1 from His6/TEV cleavage site fused human BRD4 (residues N44 to E168) expressed in Escherichia coli BL21 (DE3) incubated for 60 min by Fluorescence polarization assay	B	7.17	pIC50	68	nM	IC50	J Med Chem (2022) 65: 6573-6592 [PMID:35500243]
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	7.18	pIC50	66	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	Inhibition of recombinant his tagged BRD4 BD1 (unknown origin) by Alpha screen assay	B	7.18	pIC50	65.7	nM	IC50	Eur J Med Chem (2022) 227: 113953-113953 [PMID:34731760]
ChEMBL	Inhibition of histone binding to BRD4 (unknown origin) expressed in HEK293 cells using histone H3.3 as substrate incubated for 18 to 24 hrs by nano-BRET analysis	B	7.2	pIC50	63.1	nM	IC50	ACS Med Chem Lett (2023) 14: 1231-1236 [PMID:37736196]
ChEMBL	Binding affinity to recombinant human N-terminal His-tagged BRD4 BD1 (44 to 170 residues) expressed in Escherichia coli using H4K5AcK8Ac as substrate measured after 30 mins by AlphaScreen assay	B	7.2	pIC50	62.57	nM	IC50	Bioorg Med Chem (2020) 28: 115181-115181 [PMID:31767403]
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) after 1 hr by TR-FRET assay	B	7.21	pIC50	62	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Binding affinity human BRD4 BD2 incubated for 1 hr by TR-FRET assay	B	7.21	pIC50	62	nM	IC50	J Med Chem (2022) 65: 2388-2408 [PMID:34982556]
ChEMBL	Inhibition of His6-tagged recombinant BRD4 BD1 domain (44 to 168 residues) (unknown origin) expressed in Escherichia coli BL21(DE3) incubated for 0.5 hrs by FITC-labeled JQ1 based competitive fluorescence polarization assay	B	7.21	pIC50	61	nM	IC50	Eur J Med Chem (2024) 264: 116009-116009 [PMID:38070430]
ChEMBL	Inhibition of BRD4-BD1 in human Raji cells assessed as downregulation of MYC gene expression by PCR method	B	7.22	pIC50	60	nM	IC50	Bioorg Med Chem Lett (2018) 28: 1811-1816 [PMID:29657099]
ChEMBL	Inhibition of N-terminal His-tagged BRD4 (BD1) (unknown origin) using biotinylated tetra-acetylated histone H4 peptide after 30 mins by alpha-screen assay	B	7.22	pIC50	60	nM	IC50	J Med Chem (2018) 61: 8202-8211 [PMID:30165024]
ChEMBL	Inhibition of BRD4(1) (unknown origin) incubated for 4 hrs by (+)-JQ1 fluorescent ligand based fluorescence anisotrophy	B	7.24	pIC50	58	nM	IC50	J Med Chem (2015) 58: 1281-1297 [PMID:25559428]
ChEMBL	Binding affinity to human partial length BRD4 BD1 (N44 to E168 residues) expressed in bacterial expression system using H4K5acIC8acK12acK16ac-biotinylated peptide as substrate preincubated for 30 mins followed by substrate addition by AlphaScreen assay	B	7.25	pIC50	56.3	nM	IC50	J Med Chem (2023) 66: 913-933 [PMID:36577036]
ChEMBL	Inhibition of BRD4 D1 (unknown origin) by alphascreen assay	B	7.29	pIC50	51	nM	IC50	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Displacement of (+)-JQ1 from 6H-Thr BRD4 Y97A mutant BD2 (unknown origin) after 30 mins by TR-FRET assay	B	7.3	pIC50	50.12	nM	IC50	J Med Chem (2018) 61: 4317-4334 [PMID:29656650]
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) by TR-FRET assay	B	7.3	pIC50	50.12	nM	IC50	ACS Med Chem Lett (2023) 14: 1231-1236 [PMID:37736196]
ChEMBL	Inhibitory Activity Assay: The assays of BRD4 (D1) and BRD4 (D2) were conducted in a 384-well polystyrene plate. The test compounds were first serially diluted in DMSO and the test compound/DMSO were transferred to 384-well plates. The final concentration of DMSO in the assay was 0.1%. 2 volumes of protein/peptide mixture were added into 384-well plates, then 2 volumes of assay mixture were added, and shake for 30 s. The plate was incubated at room temperature for 2 h. Then the HTRF signal on EnVision was readed.	B	7.3	pIC50	50	nM	IC50	US-11466034-B2. Compound functioning as bromodomain protein inhibitor, and composition (2022)
ChEMBL	Inhibition of N-terminal His-tagged BRD4 (BD1/BD2) (unknown origin) using biotinylated tetra-acetylated histone H4 peptide after 30 mins by alpha-screen assay	B	7.3	pIC50	50	nM	IC50	J Med Chem (2018) 61: 8202-8211 [PMID:30165024]
ChEMBL	Inhibition of BRD4 (unknown origin) by alpha screen assay	B	7.31	pIC50	49	nM	IC50	ACS Med Chem Lett (2024) 15: 107-115 [PMID:38229743]
ChEMBL	Displacement of FITC-conjugated JQ1 from wildtype BRD4 BD1 (unknown origin) expressed in Escherichia coli BL21-DE3 rosetta cells incubated for 15 mins by competitive fluorescence polarization assay	B	7.31	pIC50	49	nM	IC50	J Med Chem (2021) 64: 11637-11650 [PMID:34279939]
ChEMBL	Inhibition of BRD4 (unknown origin) by ELISA	B	7.32	pIC50	48.12	nM	IC50	Bioorg Med Chem Lett (2021) 31: 127671-127671 [PMID:33229136]
ChEMBL	Inhibition of BRD4 (unknown origin) incubated for 30 mins by ELISA method	B	7.32	pIC50	48.12	nM	IC50	Bioorg Med Chem Lett (2021) 38: 127829-127829 [PMID:33685790]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) by TR-FRET assay	B	7.32	pIC50	48	nM	IC50	Eur J Med Chem (2023) 251: 115246-115246 [PMID:36898329]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD4 bromodomain 1 (44 to 168 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.33	pIC50	46.7	nM	IC50	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant human N-terminal His6-tagged BRD4 bromodomain 1 (44 to 168 residues) expressed in Rosetta2 DE3 cells after 30 mins by Flourescence polarization assay	B	7.33	pIC50	46.7	nM	IC50	J Med Chem (2017) 60: 3887-3901 [PMID:28463487]
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) incubated for 2 hrs by TR-FRET assay	B	7.36	pIC50	44	nM	IC50	J Med Chem (2021) 64: 18025-18053 [PMID:34908415]
ChEMBL	Acetyl-Histone Binding Assay: Assays were performed with minor modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, and 0.01% w/v Tween 20 at pH 7.5 and allowed to equilibrate to room temperature prior to addition to plates. After addition of Alpha beads to master solutions, all subsequent steps were performed in low light conditions. A 2x solution of components with final concentrations of BRD4.1 at 80 nM, Ni-coated Acceptor Bead at 25 ug/ml, and 80 nM biotinylated H4-tetra acetyl was added in 10 uL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Biotinylated peptide for BRD4.1 was synthesized in-house on a CEM Liberty 9008005 microwave peptide synthesizer: H4-tetra acetyl, biotin-PEG2-SGRGKacGGKacGLGKacGGAKacRHRK-COOH. Addition to wells was performed with either a multichannel pipet (for optimization experiments) or a Biotek EL406 liquid handler. After a 1000-rpm spin-down for 1 minute, 100 nL of the solutions of the compounds of the invention from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (25 ug/ml final) were added as with previous solution in a 2x, 10 uL volume. Following this addition, the plates were sealed with foil to block light exposure and to prevent evaporation. The plates were spun down again at 1000 rpm for 1 minute. Next, the plates were incubated in the room with the plate reader (for temperature equilibration) for 1.5 hour prior to reading the assay. AlphaScreen measurements were performed on an Envision 2104 (PerkinElmer, USA) utilizing the manufacturer's protocol.	B	7.36	pIC50	44	nM	IC50	US-9695172-B2. Diazepane derivatives and uses thereof (2017)
ChEMBL	Displacement of tetra-acetylated histone H4 peptide (1-21) from recombinant human N-terminal His-tagged BRD4 BD1 (44 to 170 residues) expressed in Escherichia coli incubated for 30 mins followed by further incubation with tetra-acetylated histone H4 peptide (1-21) for 30 mins by Alphascreen assay	B	7.37	pIC50	42.24	nM	IC50	J Med Chem (2019) 62: 11080-11107 [PMID:31789032]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant N-terminal His6-tagged BRD4 bromodomain 2 (333 to 460 residues) (unknown origin) expressed in Escherichia coli Rosetta2 DE3 after 30 mins by fluorescence polarization assay	B	7.37	pIC50	42.2	nM	IC50	J Med Chem (2018) 61: 462-481 [PMID:28339196]
ChEMBL	Inhibition of FAM-labeled ZBA248 binding to recombinant human N-terminal His6-tagged BRD4 bromodomain 2 (333 to 460 residues) expressed in Rosetta2 DE3 cells after 30 mins by Flourescence polarization assay	B	7.37	pIC50	42.2	nM	IC50	J Med Chem (2017) 60: 3887-3901 [PMID:28463487]
ChEMBL	Inhibition of BRD4-BD1 (unknown origin) preincubated for 15 mins followed by substrate addition and measured after 1 hr	B	7.38	pIC50	42	nM	IC50	Bioorg Med Chem (2021) 39: 116133-116133 [PMID:33862375]
ChEMBL	Inhibition of BRD4 bromodomain1 (unknown origin) using peptide histone H4 (SGRGACKGGACKGLGAC-KGGAACKRHGSGSK-biotin) as substrate incubated for 15 mins followed by substrate addition measured after 1 hr by alphascreen assay	B	7.38	pIC50	42	nM	IC50	ACS Med Chem Lett (2020) 11: 2174-2181 [PMID:33214826]
ChEMBL	Inhibition of N-terminal His-tagged BRD4 (BD2) (unknown origin) using biotinylated tetra-acetylated histone H4 peptide after 30 mins by alpha-screen assay	B	7.4	pIC50	40	nM	IC50	J Med Chem (2018) 61: 8202-8211 [PMID:30165024]
ChEMBL	Inhibition of recombinant human His-tagged BRD4 bromodomain 1/2 (2 to 1362 residues) expressed in s9f insect cells using biotinylated H4(1 to 21)K5/8/12/16Ac peptide as substrate preincubated for 30 mins followed by substrate addition and measured after 30 mins by Alpha screen assay	B	7.4	pIC50	40	nM	IC50	Eur J Med Chem (2019) 179: 502-514 [PMID:31276895]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) by TR-FRET assay	B	7.4	pIC50	39.81	nM	IC50	ACS Med Chem Lett (2023) 14: 1231-1236 [PMID:37736196]
ChEMBL	Displacement of (+)-JQ1 from 6H-Thr BRD4 Y390A mutant BD1 (unknown origin) after 30 mins by TR-FRET assay	B	7.4	pIC50	39.81	nM	IC50	J Med Chem (2018) 61: 4317-4334 [PMID:29656650]
ChEMBL	in vitro kinase inhibition assay: Inhibitory activity can be determined routinely using known methods and also from commercial vendors offering this service for kinases and bromodomain proteins. For example, in vitro kinase inhibition (e.g., PI3K inhibition) can be detected by a standard kinase inhibition assay using labeled ATP to determine if a test compound inhibits the transfer of phosphate from ATP to the kinase substrate. In vivo, PI3K inhibition can be determined from target tissue biopsies by standard tissue processing to disrupt cells and then performing Western Blot analysis to determine the presence or absence of pAKT (substrate of PI3K) relative to a control sample. The activity of a compound of the invention as an inhibitor of a bromodomain-containing protein, such as a BET protein, such as BRD2, BRD3, BRD4, and/or BRDT, or an isoform or mutant thereof, may be determined in vitro, in vivo, or in a cell line. In vitro assays include assays that determine inhibition of bromodomain-containing proteins. Alternatively, inhibitor binding may be determined by running a competition experiment where a provided compound is incubated with a bromodomain-containing protein, such as a BET protein bound to known ligands, labeled or unlabeled. For example, bromodomain inhibition can be determined in vitro using Alpha Screen Technology (http://www.reactionbiology.com/webapps/site/NewsPDFs/Bromodomain%20Assay%20Platform%20for%20Drug%20Screening%20and%20Discovery.pdf). In vivo bromodomain inhibition can be determined indirectly by evaluating the amount of protein present of proteins whose genes' transcription is influenced or controlled by the bromodomain protein, for example, the MYCN protein transcription is controlled by BRD4 (J. E. Delmore et al., Cell 2011, 146, 904-917; A. Puissant, Cancer Discov. 2013, 3, 308-323). Bromodomain inhibition may also be predicted by in silico modeling as described below in the Examples.	B	7.41	pIC50	39.1	nM	IC50	US-10308662-B2. Thienopyranones as kinase and epigenetic inhibitors (2019)
ChEMBL	Binding affinity to recombinant human N-terminal His-tagged BRD4 BD2 (349 to 460 residues) expressed in Escherichia coli measured after 30 mins by AlphaScreen assay	B	7.41	pIC50	39.09	nM	IC50	Bioorg Med Chem (2020) 28: 115181-115181 [PMID:31767403]
ChEMBL	Displacement of FAM-labeled ZBA248 from BRD4 BD2 (333 to 460 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells after 30 mins by fluorescence polarization assay	B	7.45	pIC50	35.7	nM	IC50	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Inhibition of BRD4-BD2 domain (unknown origin) assessed as inhibition of tetra-acetylated Histone H4 peptide binding to BRD4 by ALPHA-screen assay	B	7.48	pIC50	33	nM	IC50	Eur J Med Chem (2020) 207: 112750-112750 [PMID:32871345]
ChEMBL	Inhibition of human recombinant N-terminal 6his-tagged BRD4 expressed in Escherichia coli BL21(DE3) using H4 peptide as substrate by alphascreen assay	B	7.48	pIC50	33	nM	IC50	Bioorg Med Chem Lett (2019) 29: 126577-126577 [PMID:31421967]
ChEMBL	Selectivity interaction (AlphaScreen) EUB0000330a BRD4	B	7.48	pIC50	33	nM	IC50	Selectivity Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Inhibition of human His-tagged BRD4 BD2 domain using H-SGRGK(Ac)GGK(Ac)GLGK-(Ac)GGAK(Ac)RHRK(Biotin)-OH as substrate preincubated for 30 mins followed by substrate addition measured after 30 mins by AlphaScreen assay	B	7.48	pIC50	33	nM	IC50	Eur J Med Chem (2020) 208: 112780-112780 [PMID:32883643]
ChEMBL	Inhibition of human BRD4 BD2 by TR-FRET assay	B	7.48	pIC50	33	nM	IC50	J Med Chem (2024) 67: 922-951 [PMID:38214982]
ChEMBL	Inhibition of recombinant human His6-tagged BRD4 bromodomain 2 expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells preincubated for 30 mins followed by HSGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH peptide substrate addition after 30 mins by alphascreen assay	B	7.48	pIC50	33	nM	IC50	J Med Chem (2017) 60: 4533-4558 [PMID:28195723]
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) by alphascreen assay	B	7.48	pIC50	33	nM	IC50	Bioorg Med Chem (2024) 106: 117752-117752 [PMID:38749341]
ChEMBL	Inhibition of BRD4 (unknown origin)	B	7.48	pIC50	33	nM	IC50	Bioorg Med Chem Lett (2017) 27: 4051-4055 [PMID:28765013]
ChEMBL	Inhibition of BRD4 bromodomain 2 (unknown origin)-acetylated histone H4 interaction by luminescence proximity homogeneous assay	B	7.48	pIC50	33	nM	IC50	Medchemcomm (2012) 3: 123-134
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) by Alpha screen assay	B	7.48	pIC50	33	nM	IC50	Eur J Med Chem (2022) 227: 113953-113953 [PMID:34731760]
ChEMBL	Inhibition of His tagged human BRD4 BD2 expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells using HSGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH peptide as substrate pretreated for 30 mins followed by substrate addition and measured after 30 mins by Alphascreen analysis	B	7.48	pIC50	33	nM	IC50	Eur J Med Chem (2022) 238: 114423-114423 [PMID:35544982]
ChEMBL	Inhibition of human C-terminal BRD4 bromodomain expressed in Escherichia coli BL21(DE3) after 30 mins by luminescence proximity homogeneous assay	B	7.48	pIC50	33	nM	IC50	J Med Chem (2012) 55: 587-596 [PMID:22136469]
ChEMBL	Displacement of acetylated histone peptide from BRD4-BD2 by luminescence proximity homogenous assay	B	7.48	pIC50	33	nM	IC50	Bioorg Med Chem (2012) 20: 1887-1892 [PMID:22316554]
ChEMBL	Inhibition of BRD4-BD1 (unknown origin) incubated for 1 hr by AlphaScreen biotin-JQ1 competition assay	B	7.49	pIC50	32	nM	IC50	J Med Chem (2018) 61: 7785-7795 [PMID:30125504]
ChEMBL	Inhibition of N-terminal his6-tagged human BRD4 BD1 (44 to 168 residues) expressed in Escherichia coli Rossetta BL21 (DE3) cells incubated for 15 mins by TR-FRET assay	B	7.52	pIC50	30.3	nM	IC50	J Med Chem (2024) 67: 2712-2731 [PMID:38295759]
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) by Alphascreen assay	B	7.52	pIC50	30	nM	IC50	J Med Chem (2024) 67: 18606-18628 [PMID:39356741]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	7.52	pIC50	30	nM	IC50	Eur J Med Chem (2024) 271: 116444-116444 [PMID:38691889]
ChEMBL	Inhibition of human N-terminal 6His-tagged BRD4 BD2 (351 to 457 residues) expressed in Escherichia coli BL21-CodonPlus (DE3) cells using biotinylated H4 peptide as substrate preincubated for 30 mins followed by substrate addition and measured after 30 mins by AlphaScreen assay	B	7.53	pIC50	29.4	nM	IC50	Eur J Med Chem (2023) 247: 115023-115023 [PMID:36566713]
ChEMBL	Binding Assay: ssays were performed as described previously with minor modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 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. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150-0 μM and 4 μl transferred to low-volume 384-well plates (PROXIPLATE-384 Plus, PerkinElmer, USA), followed by 4 μl of HIS-tagged protein (BRD4/1, 250 nM, BRD4/2 and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 μl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2), and BRDT: H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH; peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH (Cambridge Research Biochemicals, UK)]. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 μl of streptavidin-coated donor beads (25 μg/ml) and 4 μl nickel chelate acceptor beads (25 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 and are given as the final concentration of compound in the 20 μl reaction volume.	B	7.54	pIC50	29	nM	IC50	US-10881668-B2. Acetamide thienotriazolodiazepines and uses thereof (2021)
ChEMBL	Inhibition of BRD4 (unknown origin)	B	7.54	pIC50	29	nM	IC50	Eur J Med Chem (2023) 262: 115879-115879 [PMID:37875056]
ChEMBL	Inhibition of recombinant BRD4-BD1 (unknown origin) by TR-FRET assay	B	7.54	pIC50	29	nM	IC50	Bioorg Med Chem Lett (2021) 31: 127671-127671 [PMID:33229136]
ChEMBL	Inhibition of recombinant BRD4 BD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	7.54	pIC50	29	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
ChEMBL	Displacement of FAM-labeled ZBA248 from BRD4 BD1 (44 to 168 amino acid residues) (unknown origin) expressed in Rosetta2 DE3 cells after 30 mins by fluorescence polarization assay	B	7.54	pIC50	28.7	nM	IC50	J Med Chem (2015) 58: 4927-4939 [PMID:26080064]
ChEMBL	Displacement of 5-FITC labelled (+)-JQ1 from His6-tagged human BRD4 bromodomain 1 expressed in Escherichia coli BL21(DE3) )-codon plus-RIL cells incubated for 4 hrs in dark condition by fluorescence anisotropy binding assay	B	7.55	pIC50	28.5	nM	IC50	J Med Chem (2019) 62: 8642-8663 [PMID:31490070]
ChEMBL	Displacement of FITC-JQ1 from His6-tagged BRD4 bromodomain-1 (unknown origin) expressed in Escherichia coli BL21(DE3) after 4 hrs by fluorescence anisotropy method	B	7.55	pIC50	28.5	nM	IC50	Eur J Med Chem (2018) 150: 156-175 [PMID:29525435]
ChEMBL	Inhibition of recombinant human GST-tagged BRD4 BD1 (44 to 168 residues) expressed in Escherichia coli incubated for 60 mins by alphascreen assay	B	7.55	pIC50	27.9	nM	IC50	Bioorg Med Chem (2020) 28: 115228-115228 [PMID:31813613]
ChEMBL	in vitro kinase inhibition assay: Inhibitory activity can be determined routinely using known methods and also from commercial vendors offering this service for kinases and bromodomain proteins. For example, in vitro kinase inhibition (e.g., PI3K inhibition) can be detected by a standard kinase inhibition assay using labeled ATP to determine if a test compound inhibits the transfer of phosphate from ATP to the kinase substrate. In vivo, PI3K inhibition can be determined from target tissue biopsies by standard tissue processing to disrupt cells and then performing Western Blot analysis to determine the presence or absence of pAKT (substrate of PI3K) relative to a control sample. The activity of a compound of the invention as an inhibitor of a bromodomain-containing protein, such as a BET protein, such as BRD2, BRD3, BRD4, and/or BRDT, or an isoform or mutant thereof, may be determined in vitro, in vivo, or in a cell line. In vitro assays include assays that determine inhibition of bromodomain-containing proteins. Alternatively, inhibitor binding may be determined by running a competition experiment where a provided compound is incubated with a bromodomain-containing protein, such as a BET protein bound to known ligands, labeled or unlabeled. For example, bromodomain inhibition can be determined in vitro using Alpha Screen Technology (http://www.reactionbiology.com/webapps/site/NewsPDFs/Bromodomain%20Assay%20Platform%20for%20Drug%20Screening%20and%20Discovery.pdf). In vivo bromodomain inhibition can be determined indirectly by evaluating the amount of protein present of proteins whose genes' transcription is influenced or controlled by the bromodomain protein, for example, the MYCN protein transcription is controlled by BRD4 (J. E. Delmore et al., Cell 2011, 146, 904-917; A. Puissant, Cancer Discov. 2013, 3, 308-323). Bromodomain inhibition may also be predicted by in silico modeling as described below in the Examples.	B	7.57	pIC50	27.1	nM	IC50	US-10308662-B2. Thienopyranones as kinase and epigenetic inhibitors (2019)
ChEMBL	Fluorescence Polarization Assay: Expression and purification of the BRD4(I) recognition domain: colonies of newly transformed plasmid DNA from E. coli BL21(DE3)-condon plus-RIL cells were cultivated in 50 mL of Terrific Broth medium containing 50 μg/mL kanamycin and 34 μg/mL chloramphenicol at 37° C. overnight (starting culture). The starting culture was then diluted 100-fold in 1 L of fresh TB medium and the cells were grown at 37° C. to an optical density of about 0.8 at and then the temperature was lowered to 16° C. When the system was equilibrated at 16° C., the optical density at was approximately 1.2, and protein expression was induced with 0.2 mmol of isopropyl-β-D-thiogalactopyranoside (IPTG) overnight at 16° C. Bacteria were harvested by centrifugation (4000×g, 20 minutes, 4° C.) and stored as a pellet at −80° C. The cells expressing His 6-tagged protein was resuspend in lysis buffer [50 mmol 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 25° C., pH 7.5, 500 mmol NaCl, 10 mmol imidazole, 5% glycerol and freshly added 0.5 mmol of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 1 mmol of phenylmethanesulfonyl fluoride (PMSF)] and lysed at 4° C. using JN 3000PLUS high pressure homogenizer (JNBIO-Guangzhou, China). The lysate was clarified by centrifugation (12,000×g for 1 hour at 4° C.) and applied to a nickel-nitriloacetate agarose column. The column was washed once with 50 mL of wash buffer containing 30 mmol of imidazole. The protein was eluted using imidazole in an elution buffer in a stepwise elution (100-250 mmol imidazole in 50 mmol HEPES, 25° C., pH 7.5, 500 mmol NaCl, 5% glycerol). All fractions were collected and monitored by SDS-polyacrylamide gel electrophoresis (Bio-Rad Criterion TM Precast Gels, 4-12% Bis-Tris, 1.0 mm, from Bio-Rad, CA). After 1 mmol of dithiothreitol (DTT) was added, the eluted proteins were treated with tobacco plaque virus (TEV) protease overnight at 4° C. to remove the His6 tag. The protein was concentrated and further purified by size exclusion chromatography on a Superdex 75 16/60 HiLoad gel filtration column. The samples were monitored by SDS-polyacrylamide gel electrophoresis and concentrated to 8-10 mg/mL with gel filtration buffer, 10 mmol Hepes pH 7.5, 500 mM NaCl, 1 mmol DTT, and used for protein binding assays and crystallization.	B	7.57	pIC50	27	nM	IC50	US-11078188-B2. Dihydroquinoxaline bromodomain recognition protein inhibitor, preparation method and use thereof (2021)
ChEMBL	Displacement of tetra-acetylated histone H4 peptide (1-21) from recombinant human N-terminal His-tagged BRD4 BD2 (349 to 460 residues) expressed in Escherichia coli incubated for 30 mins followed by further incubation with tetra-acetylated histone H4 peptide (1-21) for 30 mins by Alphascreen assay	B	7.61	pIC50	24.71	nM	IC50	J Med Chem (2019) 62: 11080-11107 [PMID:31789032]
ChEMBL	Displacement of biotinylated tetra-acetylated histone H4 from human his-tagged BRD4 bromodomain1 by alphascreen assay	B	7.62	pIC50	24	nM	IC50	J Med Chem (2016) 59: 1271-1298 [PMID:26572217]
ChEMBL	Inhibition of His-FLAG-tagged BRD4 binding domain1 (unknown origin) binding to H4-TetraAc-biotin peptide after 20 mins by AlphaLISA	B	7.62	pIC50	24	nM	IC50	ACS Med Chem Lett (2013) 4: 835-840 [PMID:24900758]
ChEMBL	Inhibition of His/FLAG-tagged BRD4 BD1 (unknown origin) using histone H4-TetraAcetylated-biotin peptide as substrate incubated for 20 mins by AlphaLISA assay	B	7.62	pIC50	24	nM	IC50	ACS Med Chem Lett (2016) 7: 145-150 [PMID:26985289]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) by TR-FRET assay	B	7.64	pIC50	22.9	nM	IC50	Bioorg Med Chem (2023) 78: 117152-117152 [PMID:36599264]
ChEMBL	Inhibition of human recombinant His-tagged BRD4 BD1 domain using H-SGRGK(Ac)GGK(Ac)GLGK-(Ac)GGAK(Ac)RHRK(Biotin)-OH as substrate preincubated with enzyme for 30 mins followed by substrate addition measured after 30 mins by AlphaScreen assay	B	7.66	pIC50	22	nM	IC50	J Med Chem (2020) 63: 3678-3700 [PMID:32153186]
ChEMBL	Binding affinity to human BRD4 BD1 (44 to 168 residues) by fluorescence polarization assay	B	7.66	pIC50	22	nM	IC50	J Med Chem (2018) 61: 6685-6704 [PMID:30019901]
ChEMBL	Inhibition of BRD4 bromodomain 1 (unknown origin) by Alphascreen based method	B	7.66	pIC50	22	nM	IC50	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Inhibition of human BRD4 BD2 (342 to 460 residues) at 30 uM preincubated for 30 mins followed by substrate addition measured after 30 mins by TR-FRET assay	B	7.68	pIC50	21	nM	IC50	Eur J Med Chem (2016) 121: 294-299 [PMID:27266999]
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	7.68	pIC50	21	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	7.72	pIC50	19	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)
ChEMBL	Alpha Assay: BRD4(1): Assays were performed as described previously with minor modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 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. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150-0 μM and 4 μl transferred to low-volume 384-well plates (ProxiPlate™-384 Plus, PerkinElmer, USA), followed by 4 μl of HIS-tagged protein (BRD4/1, 250 nM, BRD4/2 and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 μl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2): H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH (SEQ ID NO: 5); peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH (SEQ ID NO: 6) (Cambridge Research Biochemicals, UK)]. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 μl of streptavidin-coated donor beads (25 μg/ml) and 4 μl nickel chelate acceptor beads (25 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 and are given as the final concentration of compound in the 20 μl reaction volume.	B	7.74	pIC50	18	nM	IC50	US-9320741-B2. Compositions and methods for treating neoplasia, inflammatory disease and other disorders (2016)
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) by HTRF assay	B	7.74	pIC50	18	nM	IC50	Eur J Med Chem (2024) 263: 115924-115924 [PMID:37992518]
ChEMBL	Binding Assay: All reagents were diluted in 50 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. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150-0 μM and 4 μl transferred to low-volume 384-well plates (ProxiPlate-384 Plus, PerkinElmer, USA), followed by 4 μl of HIS-tagged protein (BRD4/1, 250 nM, BRD4/2 and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 μl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2): H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH (SEQ ID NO: 5); peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH (SEQ ID NO: 6) (Cambridge Research Biochemicals, UK)]. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 μl of streptavidin-coated donor beads (25 μg/ml) and 4 μl nickel chelate acceptor beads (25 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 and are given as the final concentration of compound in the 20 μl reaction volume.	B	7.74	pIC50	18	nM	IC50	US-10407441-B2. Compositions and methods for treating neoplasia, inflammatory disease and other disorders (2019)
ChEMBL	Alpha-assay: The binding activity of lead compounds with the BRD4 site 1 was determined by Alpha-assay with a 12-point dose response curve	B	7.74	pIC50	18	nM	IC50	US-10925881-B2. Treatment of conditions associated with hyperinsulinaemia (2021)
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) by TR-FRET assay	B	7.77	pIC50	17	nM	IC50	Bioorg Med Chem Lett (2024) 109: 129848-129848 [PMID:38876176]
ChEMBL	Inhibitory Activity Assay: The assays of BRD4 (D1) and BRD4 (D2) were conducted in a 384-well polystyrene plate. The test compounds were first serially diluted in DMSO and the test compound/DMSO were transferred to 384-well plates. The final concentration of DMSO in the assay was 0.1%. 2 volumes of protein/peptide mixture were added into 384-well plates, then 2 volumes of assay mixture were added, and shake for 30 s. The plate was incubated at room temperature for 2 h. Then the HTRF signal on EnVision was readed.	B	7.8	pIC50	16	nM	IC50	US-11466034-B2. Compound functioning as bromodomain protein inhibitor, and composition (2022)
ChEMBL	Inhibition of BRD4 BD2 (unknown origin)	B	7.82	pIC50	15	nM	IC50	Bioorg Med Chem Lett (2024) 109: 129849-129849 [PMID:38876177]
ChEMBL	Inhibition of BRD4-BD1 (unknown origin) assessed as peptide-biotin titration - BRD4(1)/histone H4 peptide interaction by HTRF/EPIgeneous assay	B	7.82	pIC50	15	nM	IC50	J Med Chem (2024) 67: 138-151 [PMID:38153295]
ChEMBL	Inhibition of C-terminal His6-tagged BRD4 BD1 (unknown origin) using H-YSGRGK(Ac)GGK(Ac)GLGK(Ac)-GGAK(Ac)RHRK-Biotin-OH as substrate incubated for 1 hrs by HTRF assay	B	7.85	pIC50	14	nM	IC50	J Med Chem (2022) 65: 5760-5799 [PMID:35333526]
ChEMBL	Binding Assay: All reagents were diluted in 50 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. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150-0 μM and 4 μl transferred to low-volume 384-well plates (ProxiPlate-384 Plus, PerkinElmer, USA), followed by 4 μl of HIS-tagged protein (BRD4/1, 250 nM, BRD4/2 and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 μl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2): H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH (SEQ ID NO: 5); peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH (SEQ ID NO: 6) (Cambridge Research Biochemicals, UK)]. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 μl of streptavidin-coated donor beads (25 μg/ml) and 4 μl nickel chelate acceptor beads (25 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 and are given as the final concentration of compound in the 20 μl reaction volume.	B	7.85	pIC50	14	nM	IC50	US-10407441-B2. Compositions and methods for treating neoplasia, inflammatory disease and other disorders (2019)
ChEMBL	Alpha Assay: BRD4(2): Assays were performed as described previously with minor modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 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. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150-0 μM and 4 μl transferred to low-volume 384-well plates (ProxiPlate™-384 Plus, PerkinElmer, USA), followed by 4 μl of HIS-tagged protein (BRD4/1, 250 nM, BRD4/2 and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 μl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2): H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH (SEQ ID NO: 5); peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH (SEQ ID NO: 6) (Cambridge Research Biochemicals, UK)]. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 μl of streptavidin-coated donor beads (25 μg/ml) and 4 μl nickel chelate acceptor beads (25 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 and are given as the final concentration of compound in the 20 μl reaction volume.	B	7.85	pIC50	14	nM	IC50	US-9320741-B2. Compositions and methods for treating neoplasia, inflammatory disease and other disorders (2016)
ChEMBL	Alpha-assay: The binding activity of lead compounds with the BRD4 site 2 was determined by Alpha-assay with a 12-point dose response curve	B	7.85	pIC50	14	nM	IC50	US-10925881-B2. Treatment of conditions associated with hyperinsulinaemia (2021)
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) by TR-FRET assay	B	7.89	pIC50	13	nM	IC50	Eur J Med Chem (2023) 251: 115246-115246 [PMID:36898329]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) preincubated for 15 mins followed by peptide addition and measured after 60 mins by TR-FRET assay	B	7.92	pIC50	12	nM	IC50	Eur J Med Chem (2020) 208: 112780-112780 [PMID:32883643]
ChEMBL	Inhibition of BRD4 BD1 (unknown origin) incubated for 15 mins by TR-FRET assay	B	8	pIC50	10	nM	IC50	Bioorg Med Chem (2024) 106: 117752-117752 [PMID:38749341]
ChEMBL	Displacement of biotinylated acetylated histone H4 peptide from human recombinant N-terminal GST-tagged BRD4 BD1 (49 to 170 residues) expressed in Escherichia coli expression system incubated for 30 mins by AlphaScreen assay	B	8.05	pIC50	9	nM	IC50	Bioorg Med Chem Lett (2022) 64: 128696-128696 [PMID:35318165]
ChEMBL	Inhibition of BRD4 bromodomain-1 (unknown origin) by europium based LANCE TR-FRET assay	B	8.1	pIC50	8	nM	IC50	Bioorg Med Chem (2015) 23: 953-959 [PMID:25678016]
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) by HTRF assay	B	8.1	pIC50	7.9	nM	IC50	Eur J Med Chem (2024) 263: 115924-115924 [PMID:37992518]
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) incubated for 15 mins by TR-FRET assay	B	8.24	pIC50	5.7	nM	IC50	Bioorg Med Chem (2024) 106: 117752-117752 [PMID:38749341]
ChEMBL	Inhibition of C-terminal His6-tagged BRD4 BD2 (unknown origin) using H-YSGRGK(Ac)GGK(Ac)GLGK(Ac)-GGAK(Ac)RHRK-Biotin-OH as substrate incubated for 1 hrs by HTRF assay	B	8.27	pIC50	5.31	nM	IC50	J Med Chem (2022) 65: 5760-5799 [PMID:35333526]
ChEMBL	Binding affinity to human partial length BRD4 BD2 (K333 to E460 residues) expressed in bacterial expression system using H4K5acIC8acK12acK16ac-biotinylated peptide as substrate preincubated for 30 mins followed by substrate addition by AlphaScreen assay	B	8.29	pIC50	5.16	nM	IC50	J Med Chem (2023) 66: 913-933 [PMID:36577036]
ChEMBL	Affinity Phenotypic Cellular interaction (Cell Proliferation (Cell TiterGlo assay Promega in NMC 11060 cells)) EUB0000330a BRD4	F	8.4	pIC50	4	nM	IC50	Affinity Phenotypic Cellular Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Affinity Phenotypic Cellular interaction (Cell Proliferation (Cell TiterGlo assay Promega in NMC 11060 cells)) EUB0000330a BRD4	F	8.4	pIC50	4	nM	IC50	Affinity Phenotypic Cellular Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Inhibition of BRD4 BD2 (unknown origin) preincubated for 15 mins followed by peptide addition and measured after 60 mins by TR-FRET assay	B	8.41	pIC50	3.9	nM	IC50	Eur J Med Chem (2020) 208: 112780-112780 [PMID:32883643]
ChEMBL	Inhibition of halo-tagged BRD4 (unknown origin) expressed in HEK293 cells incubated for 4 hrs by biosensor assay	B	5	pEC50	>10000	nM	EC50	ACS Med Chem Lett (2023) 14: 1231-1236 [PMID:37736196]
ChEMBL	Inhibition of BRD4 in human H1299 cells assessed as decrease in HPV LCR-E2-EP400-mediated transcriptional repression after 24 hrs by Bright-Glo luciferase reporter gene assay	B	6.82	pEC50	153	nM	EC50	J Med Chem (2017) 60: 8369-8384 [PMID:28949521]
ChEMBL	Inhibition of BRD4 in human MX1 cells assessed as decrease in cell proliferation after 3 days by Celltiter-Glo assay	B	6.84	pEC50	144	nM	EC50	Medchemcomm (2019) 10: 974-984 [PMID:31303996]
ChEMBL	Inhibition of BRD4 in human H1229 cells transfected with HPV-E2 assessed as transrepression of HPV-long control region by luciferase reporter assay	B	7.19	pEC50	64	nM	EC50	Medchemcomm (2019) 10: 974-984 [PMID:31303996]
bromodomain containing 4/Bromodomain-containing protein 4 in Mouse (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL3085619] [GtoPdb: 1945] [UniProtKB: Q9ESU6]
ChEMBL	Inhibition of BRD4 in mouse primary skin-derived precursors (Nf1-/-, P53-/-) assessed as reduction in cell viability incubated for 72 hrs by SRB assay	B	6.4	pIC50	<400	nM	IC50	PLoS One (2017) 12: 183155-183155 [PMID:28813519]
ChEMBL	Inhibition of BRD4 in mouse MPNST cells assessed as reduction in cell viability incubated for 72 hrs by SRB assay	B	6.4	pIC50	<400	nM	IC50	PLoS One (2017) 12: 183155-183155 [PMID:28813519]
bromodomain containing 9/Bromodomain-containing protein 9 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL3108640] [GtoPdb: 2728] [UniProtKB: Q9H8M2]
ChEMBL	Binding affinity to BRD9 (unknown origin) by TR-FRET assay	B	5	pIC50	>10000	nM	IC50	J Med Chem (2024) 67: 14125-14154 [PMID:39132814]
bromodomain testis associated/Bromodomain testis-specific protein in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL1795185] [GtoPdb: 2729] [UniProtKB: Q58F21]
ChEMBL	Binding affinity to recombinant human N-terminal hexaHis-tagged BRDT expressed in Escherichia coli BL21 (DE3) cells by MST assay	B	6.59	pKd	260	nM	Kd	J Med Chem (2021) 64: 15772-15786 [PMID:34710325]
ChEMBL	Binding affinity to recombinant BRDT BD2 (unknown origin) incubated for 1 hr by TR-FRET assay	B	6.66	pKd	217	nM	Kd	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Affinity Biochemical interaction (Isothermal titration calorimetry (ITC)) EUB0000330a BRDT	B	6.72	pKd	190.1	nM	Kd	Affinity Biochemical Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Binding affinity to BRDT-BD1 by isothermal titration calorimetry	B	6.72	pKd	190.1	nM	Kd	Bioorg Med Chem (2012) 20: 1887-1892 [PMID:22316554]
ChEMBL	Binding affinity to his tagged BRDT bromodomain (unknown origin) assessed as dissociation constant by SPR method	B	6.73	pKd	188	nM	Kd	ACS Med Chem Lett (2024) 15: 107-115 [PMID:38229743]
ChEMBL	Binding affinity to recombinant human N-terminal hexaHis-tagged BRDT expressed in Escherichia coli BL21 (DE3) cells by ITC analysis	B	6.73	pKd	188	nM	Kd	J Med Chem (2021) 64: 15772-15786 [PMID:34710325]
ChEMBL	Binding affinity to recombinant BRDT BD1 (unknown origin) incubated for 1 hr by TR-FRET assay	B	6.74	pKd	183	nM	Kd	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Binding affinity to recombinant human N-terminal hexaHis-tagged BRDT expressed in Escherichia coli BL21 (DE3) cells by qPCR assay	B	7.66	pKd	22	nM	Kd	J Med Chem (2021) 64: 15772-15786 [PMID:34710325]
ChEMBL	Inhibition of recombinant BRDT BD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.23	pIC50	588	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
ChEMBL	Inhibition of recombinant BRDT BD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.27	pIC50	534	nM	IC50	J Med Chem (2023) 66: 1239-1253 [PMID:36622852]
ChEMBL	Binding affinity to human partial length BRDT BD1 (N21 to E137 residues) expressed in bacterial expression system using H4K5acIC8acK12acK16ac-biotinylated peptide as substrate preincubated for 30 mins followed by substrate addition by AlphaScreen assay	B	6.29	pIC50	510	nM	IC50	J Med Chem (2023) 66: 913-933 [PMID:36577036]
ChEMBL	Inhibition of BRDT BD1 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.36	pIC50	433	nM	IC50	Eur J Med Chem (2024) 271: 116444-116444 [PMID:38691889]
ChEMBL	Inhibition of recombinant BRDT BD2 (unknown origin) incubated for 120 mins by TR-FRET assay	B	6.49	pIC50	325	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
ChEMBL	Binding affinity BRDT BD2 (unknown origin) incubated for 1 hr by TR-FRET assay	B	6.65	pIC50	224	nM	IC50	J Med Chem (2022) 65: 2388-2408 [PMID:34982556]
ChEMBL	Inhibition of BRDT BD2 (unknown origin) after 1 hr by TR-FRET assay	B	6.66	pIC50	217	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Inhibition of BRDT BD1 (unknown origin) incubated for 2 hrs by TR-FRET assay	B	6.68	pIC50	207	nM	IC50	J Med Chem (2021) 64: 18025-18053 [PMID:34908415]
ChEMBL	Inhibition of BRDT BD2 (unknown origin) incubated for 2 hrs by TR-FRET assay	B	6.73	pIC50	185	nM	IC50	J Med Chem (2021) 64: 18025-18053 [PMID:34908415]
ChEMBL	Inhibition of BRDT BD1 (unknown origin) after 1 hr by TR-FRET assay	B	6.74	pIC50	183	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Binding affinity BRDT BD1 (unknown origin) incubated for 1 hr by TR-FRET assay	B	6.75	pIC50	178	nM	IC50	J Med Chem (2022) 65: 2388-2408 [PMID:34982556]
ChEMBL	Acetyl-Histone Binding Assay: Assays were performed with minor modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, and 0.01% w/v Tween 20 at pH 7.5 and allowed to equilibrate to room temperature prior to addition to plates. After addition of Alpha beads to master solutions, all subsequent steps were performed in low light conditions. A 2x solution of components with final concentrations of BRD4.1 at 80 nM, Ni-coated Acceptor Bead at 25 ug/ml, and 80 nM biotinylated H4-tetra acetyl was added in 10 uL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). Biotinylated peptide for BRD4.1 was synthesized in-house on a CEM Liberty 9008005 microwave peptide synthesizer: H4-tetra acetyl, biotin-PEG2-SGRGKacGGKacGLGKacGGAKacRHRK-COOH. Addition to wells was performed with either a multichannel pipet (for optimization experiments) or a Biotek EL406 liquid handler. After a 1000-rpm spin-down for 1 minute, 100 nL of the solutions of the compounds of the invention from stock plates were added by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (25 ug/ml final) were added as with previous solution in a 2x, 10 uL volume. Following this addition, the plates were sealed with foil to block light exposure and to prevent evaporation. The plates were spun down again at 1000 rpm for 1 minute. Next, the plates were incubated in the room with the plate reader (for temperature equilibration) for 1.5 hour prior to reading the assay. AlphaScreen measurements were performed on an Envision 2104 (PerkinElmer, USA) utilizing the manufacturer's protocol.	B	6.83	pIC50	147	nM	IC50	US-9695172-B2. Diazepane derivatives and uses thereof (2017)
ChEMBL	Inhibition of BRDT bromodomain 1 (unknown origin) by Alphascreen based method	B	6.87	pIC50	134	nM	IC50	Eur J Med Chem (2023) 246: 114970-114970 [PMID:36470106]
ChEMBL	Inhibition of human recombinant His-tagged BRDT BD1 domain expressed in Escherichia coli BL21(DE3)-R3-pRARE2 cells by AlphaScreen assay	B	6.87	pIC50	134	nM	IC50	J Med Chem (2020) 63: 3678-3700 [PMID:32153186]
ChEMBL	Binding affinity to BRDT bromodomain (unknown origin) by alpha screen assay	B	6.91	pIC50	124	nM	IC50	ACS Med Chem Lett (2024) 15: 107-115 [PMID:38229743]
ChEMBL	Inhibition of BRDT D1 (unknown origin) by competitive fluorescence anisotropy assay	B	6.92	pIC50	120	nM	IC50	J Med Chem (2022) 65: 2342-2360 [PMID:35007061]
ChEMBL	Inhibition of BRDT-BD1 (unknown origin) incubated for 1 hr by AlphaScreen biotin-JQ1 competition assay	B	6.99	pIC50	103	nM	IC50	J Med Chem (2018) 61: 7785-7795 [PMID:30125504]
ChEMBL	Binding Assay: ssays were performed as described previously with minor modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 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. A 24-point 1:2 serial dilution of the ligands was prepared over the range of 150-0 μM and 4 μl transferred to low-volume 384-well plates (PROXIPLATE-384 Plus, PerkinElmer, USA), followed by 4 μl of HIS-tagged protein (BRD4/1, 250 nM, BRD4/2 and CREBBP, 2000 nM). Plates were sealed and incubated at room temperature for 30 minutes, before the addition of 4 μl of biotinylated peptide at equimolar concentration to the protein [peptide for BRD4(1) & BRD4(2), and BRDT: H4K5acK8acK12acK16ac, H-SGRGK(Ac)GGK(Ac)GLGK(Ac)GGAK(Ac)RHRK(Biotin)-OH; peptide for CREBBP: H3K36ac, Biotin-KSAPATGGVK(Ac)KPHRYRPGT-OH (Cambridge Research Biochemicals, UK)]. Plates were sealed and incubated for a further 30 minutes, before the addition of 4 μl of streptavidin-coated donor beads (25 μg/ml) and 4 μl nickel chelate acceptor beads (25 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 and are given as the final concentration of compound in the 20 μl reaction volume.	B	7.11	pIC50	77	nM	IC50	US-10881668-B2. Acetamide thienotriazolodiazepines and uses thereof (2021)
ChEMBL	Inhibition of BRDT (unknown origin)	B	7.46	pIC50	35	nM	IC50	J Med Chem (2016) 59: 9305-9320 [PMID:27559828]
ChEMBL	Inhibition of BRDT-BD1 (unknown origin) by HTRF analysis	B	7.64	pIC50	23	nM	IC50	J Med Chem (2023) 66: 2646-2662 [PMID:36774555]
GtoPdb	Inhibition of H4Kac4 binding to hBRDT-BD1	-	7.96	pIC50	11	nM	IC50	Cell (2012) 150: 673-84 [PMID:22901802]
ChEMBL	Binding affinity to human partial length BRDT BD2 (K250 to E382 residues) expressed in bacterial expression system using H4K5acIC8acK12acK16ac-biotinylated peptide as substrate preincubated for 30 mins followed by substrate addition by AlphaScreen assay	B	8.26	pIC50	5.47	nM	IC50	J Med Chem (2023) 66: 913-933 [PMID:36577036]
ChEMBL	Affinity Phenotypic Cellular interaction (Cell Proliferation (Cell TiterGlo assay Promega in NMC 11060 cells)) EUB0000330a BRDT	F	8.4	pIC50	4	nM	IC50	Affinity Phenotypic Cellular Literature for EUbOPEN Chemogenomics Library wave 3
ChEMBL	Affinity Phenotypic Cellular interaction (Cell Proliferation (Cell TiterGlo assay Promega in NMC 11060 cells)) EUB0000330a BRDT	F	8.4	pIC50	4	nM	IC50	Affinity Phenotypic Cellular Literature for EUbOPEN Chemogenomics Library wave 3
bromodomain testis associated in Mouse [GtoPdb: 2729] [UniProtKB: Q91Y44]
GtoPdb	Inhibition of H4Kac4 binding to mBRDT-BD1	-	8	pIC50	10	nM	IC50	Cell (2012) 150: 673-84 [PMID:22901802]
C-C motif chemokine 2 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL1649052] [UniProtKB: P13500]
ChEMBL	Inhibition of MCP-1 in LPS-stimulated human whole blood pretreated with compound for 30 mins followed by LPS-stimulation and measured after 10 mins by microplate analysis	B	6.2	pIC50	630.96	nM	IC50	ACS Med Chem Lett (2023) 14: 1231-1236 [PMID:37736196]
ChEMBL	Inhibition of MCP-1 in LPS-stimulated human PBMC cells pretreated with compound for 30 mins followed by LPS-stimulation and measured after 10 mins by microplate analysis	B	8.2	pIC50	6.31	nM	IC50	ACS Med Chem Lett (2023) 14: 1231-1236 [PMID:37736196]
CREB binding lysine acetyltransferase/CREB-binding protein in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL5747] [GtoPdb: 2734] [UniProtKB: Q92793]
ChEMBL	Binding affinity to recombinant CBP (unknown origin) incubated for 1 hr by TR-FRET assay	B	5.02	pKd	9600	nM	Kd	J Med Chem (2020) 63: 5242-5256 [PMID:32255647]
ChEMBL	Displacement of acetylated histone peptide from CREBBP by luminescence proximity homogenous assay	B	5	pIC50	>10000	nM	IC50	Bioorg Med Chem (2012) 20: 1887-1892 [PMID:22316554]
ChEMBL	Inhibition of CBP (unknown origin) after 1 hr by TR-FRET assay	B	5.02	pIC50	9600	nM	IC50	Eur J Med Chem (2018) 151: 450-461 [PMID:29649741]
ChEMBL	Binding affinity CBP (unknown origin) incubated for 1 hr by TR-FRET assay	B	5.02	pIC50	9546	nM	IC50	J Med Chem (2022) 65: 2388-2408 [PMID:34982556]
ChEMBL	In Vitro Enzyme Inhibition Assay-CREBBP Inhibition: Determination of the IC50 for the CREBBP inhibitors disclosed herein was performed as follows: CREBBP was cloned and expressed in E. Coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CREBBP binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CREBBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20-minute incubation at room temperature, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After two hours of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.The ability of the compounds disclosed herein to inhibit CBP activity was quantified and the respective IC50 value was determined.	B	5.3	pIC50	>5000	nM	IC50	US-10202360-B2. Therapeutic compounds (2019)
ChEMBL	In Vitro CBP Inhibition: The CBP-inhibitory activity of the compounds described herein was determined by calculating the IC50. More specifically, CBP inhibitor activity was assayed as follows: CBP was cloned and expressed in E. coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CBP binding and inhibition is assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at room temp, Alpha-streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 calculated using a four parameter non-linear curve fit.	B	5.3	pIC50	>5000	nM	IC50	US-10617680-B2. Therapeutic compounds (2020)
Delta and Notch-like epidermal growth factor-related receptor in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL5291567] [UniProtKB: Q8NFT8]
ChEMBL	Inhibition of BET (unknown origin)	B	6.3	pIC50	500	nM	IC50	J Med Chem (2021) 64: 2382-2418 [PMID:33650861]
EP300 lysine acetyltransferase/Histone acetyltransferase p300 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL3784] [GtoPdb: 2735] [UniProtKB: Q09472]
ChEMBL	Inhibition of P300 (unknown orign) using acetyl-CoA as substrate preincubated for 30 mins followed by substrate addition measured after 1 hr by microplate reader analysis	B	4.71	pIC50	19400	nM	IC50	J Med Chem (2021) 64: 17413-17435 [PMID:34813314]
phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta/Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL3130] [GtoPdb: 2155] [UniProtKB: O00329]
ChEMBL	in vitro kinase inhibition assay: Inhibitory activity can be determined routinely using known methods and also from commercial vendors offering this service for kinases and bromodomain proteins. For example, in vitro kinase inhibition (e.g., PI3K inhibition) can be detected by a standard kinase inhibition assay using labeled ATP to determine if a test compound inhibits the transfer of phosphate from ATP to the kinase substrate. In vivo, PI3K inhibition can be determined from target tissue biopsies by standard tissue processing to disrupt cells and then performing Western Blot analysis to determine the presence or absence of pAKT (substrate of PI3K) relative to a control sample. The activity of a compound of the invention as an inhibitor of a bromodomain-containing protein, such as a BET protein, such as BRD2, BRD3, BRD4, and/or BRDT, or an isoform or mutant thereof, may be determined in vitro, in vivo, or in a cell line. In vitro assays include assays that determine inhibition of bromodomain-containing proteins. Alternatively, inhibitor binding may be determined by running a competition experiment where a provided compound is incubated with a bromodomain-containing protein, such as a BET protein bound to known ligands, labeled or unlabeled. For example, bromodomain inhibition can be determined in vitro using Alpha Screen Technology (http://www.reactionbiology.com/webapps/site/NewsPDFs/Bromodomain%20Assay%20Platform%20for%20Drug%20Screening%20and%20Discovery.pdf). In vivo bromodomain inhibition can be determined indirectly by evaluating the amount of protein present of proteins whose genes' transcription is influenced or controlled by the bromodomain protein, for example, the MYCN protein transcription is controlled by BRD4 (J. E. Delmore et al., Cell 2011, 146, 904-917; A. Puissant, Cancer Discov. 2013, 3, 308-323). Bromodomain inhibition may also be predicted by in silico modeling as described below in the Examples.	B	4.3	pIC50	>50000	nM	IC50	US-10308662-B2. Thienopyranones as kinase and epigenetic inhibitors (2019)
phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma/Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL3267] [GtoPdb: 2156] [UniProtKB: P48736]
ChEMBL	in vitro kinase inhibition assay: Inhibitory activity can be determined routinely using known methods and also from commercial vendors offering this service for kinases and bromodomain proteins. For example, in vitro kinase inhibition (e.g., PI3K inhibition) can be detected by a standard kinase inhibition assay using labeled ATP to determine if a test compound inhibits the transfer of phosphate from ATP to the kinase substrate. In vivo, PI3K inhibition can be determined from target tissue biopsies by standard tissue processing to disrupt cells and then performing Western Blot analysis to determine the presence or absence of pAKT (substrate of PI3K) relative to a control sample. The activity of a compound of the invention as an inhibitor of a bromodomain-containing protein, such as a BET protein, such as BRD2, BRD3, BRD4, and/or BRDT, or an isoform or mutant thereof, may be determined in vitro, in vivo, or in a cell line. In vitro assays include assays that determine inhibition of bromodomain-containing proteins. Alternatively, inhibitor binding may be determined by running a competition experiment where a provided compound is incubated with a bromodomain-containing protein, such as a BET protein bound to known ligands, labeled or unlabeled. For example, bromodomain inhibition can be determined in vitro using Alpha Screen Technology (http://www.reactionbiology.com/webapps/site/NewsPDFs/Bromodomain%20Assay%20Platform%20for%20Drug%20Screening%20and%20Discovery.pdf). In vivo bromodomain inhibition can be determined indirectly by evaluating the amount of protein present of proteins whose genes' transcription is influenced or controlled by the bromodomain protein, for example, the MYCN protein transcription is controlled by BRD4 (J. E. Delmore et al., Cell 2011, 146, 904-917; A. Puissant, Cancer Discov. 2013, 3, 308-323). Bromodomain inhibition may also be predicted by in silico modeling as described below in the Examples.	B	4.3	pIC50	>50000	nM	IC50	US-10308662-B2. Thienopyranones as kinase and epigenetic inhibitors (2019)
Janus kinase 2/Tyrosine-protein kinase JAK2 in Human (target type: SINGLE PROTEIN) [ChEMBL: CHEMBL2971] [GtoPdb: 2048] [UniProtKB: O60674]
ChEMBL	AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)	B	5	pIC50	10000	nM	IC50	US-11279703-B2. Fused pyrimidine compounds as BRD4 and JAK2 dual inhibitors and methods for use thereof (2022)

ChEMBL data shown on this page come from version 36:

Zdrazil B, Felix E, Hunter F, Manners EJ, Blackshaw J, Corbett S, de Veij M, Ioannidis H, Lopez DM, Mosquera JF, Magarinos MP, Bosc N, Arcila R, Kizilören T, Gaulton A, Bento AP, Adasme MF, Monecke P, Landrum GA, Leach AR. (2024). The ChEMBL Database in 2023: a drug discovery platform spanning multiple bioactivity data types and time periods. Nucleic Acids Res., 52(D1). DOI: 10.1093/nar/gkad1004. [EPMCID:10767899] [PMID:37933841]
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