Assay Performance
All BROMO
scan assays were validated with small molecule ligands and/or acetylated histone tail peptides. Measured Kd’s were compared to published isothermal calorimetry (ITC) values.
Contact us for a complete data validation package.
Family IV Assay Validation – BRD1
A. Ligand Affinity Measurement
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B. Selective Binding to Acetylated Histone Tail Peptide
| Bromodomain |
Histone Tail Peptide |
BROMOscan Kd (nM) |
| BRD1 |
H4(Ac)1 |
15,000 |
| BRD1 |
H42 |
>100,000 |
| 1internally discovered acetylated peptide ligand. 2corresponding non-acetylated peptide. |
BRD1 was tested in Kd mode using a known bromodomain ligand (Panel A) and acetylated/non-acetylated histone tail peptide (Panel B).
bromoKdELECT Assay Panel
Listed below are the assays currently available for screening and profiling.
| BGS ▲ | Bromodomain Protein Name | Entrez Gene Symbol |
| ATAD2A | ATPase family AAA domain-containing protein 2 | ATAD2 |
| ATAD2B | ATPase family, AAA domain containing 2B | ATAD2B |
| BAZ2A | bromodomain adjacent to zinc finger domain, 2A | BAZ2A |
| BAZ2B | bromodomain adjacent to zinc finger domain, 2B | BAZ2B |
| BRD1 | bromodomain-containing protein 1 | BRD1 |
| BRD2(1) | bromodomain-containing protein 2 isoform 1, bromodomain 1 | BRD2 |
| BRD2(1,2) | bromodomain-containing protein 2 isoform 1, bromodomains 1 and 2 | BRD2 |
| BRD2(2) | bromodomain-containing protein 2 isoform 1, bromodomain 2 | BRD2 |
| BRD3(1) | bromodomain-containing protein 3, bromodomain 1 | BRD3 |
| BRD3(1,2) | bromodomain-containing protein 3, bromodomains 1 and 2 | BRD3 |
| BRD3(2) | bromodomain-containing protein 3, bromodomain 2 | BRD3 |
| BRD4(1) | bromodomain-containing protein 4 isoform long, bromodomain 1 | BRD4 |
| BRD4(1,2) | bromodomain-containing protein 4 isoform long, bromodomains 1 and 2 | BRD4 |
| BRD4(2) | bromodomain-containing protein 4 isoform long, bromodomain 2 | BRD4 |
| BRD4(full-length,short-iso.) | bromodomain-containing protein 4 isoform short | BRD4 |
| BRD7 | bromodomain containing 7 | BRD7 |
| BRD8(1) | bromodomain containing 8, bromodomain 1 | BRD8 |
| BRD8(2) | bromodomain containing 8, bromodomain 2 | BRD8 |
| BRD9 | bromodomain-containing protein 9 isoform 1 | BRD9 |
| BRDT(1) | bromodomain testis-specific protein isoform b, bromodomain 1 | BRDT |
| BRDT(1,2) | bromodomain testis-specific protein isoform b, bromodomains 1 and 2 | BRDT |
| BRDT(2) | bromodomain testis-specific protein isoform b, bromodomain 2 | BRDT |
| BRPF1 | bromodomain and PHD finger containing, 1 | BRPF1 |
| BRPF3 | bromodomain and PHD finger containing, 3 | BRPF3 |
| CECR2 | cat eye syndrome chromosome region, candidate 2 | CECR2 |
| CREBBP | CREB binding protein | CREBBP |
| EP300 | E1A binding protein p300 | EP300 |
| FALZ | nucleosome-remodeling factor subunit BPTF isoform 1 | BPTF |
| GCN5L2 | K(lysine) acetyltransferase 2A | KAT2A |
| PBRM1(2) | polybromo 1, bromodomain 2 | PBRM1 |
| PBRM1(5) | polybromo 1, bromodomain 5 | PBRM1 |
| PCAF | K(lysine) acetyltransferase 2B | KAT2B |
| SMARCA2 | SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 | SMARCA2 |
| SMARCA4 | SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 | SMARCA4 |
| TAF1(2) | TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 250kDa, bromodomain 2 | TAF1 |
| TAF1L(2) | TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 210kDa-like, bromodomain 2 | TAF1L |
| TRIM24(Bromo.) | tripartite motif containing 24 | TRIM24 |
| TRIM24(PHD,Bromo.) | tripartite motif containing 24 | TRIM24 |
| TRIM33(PHD,Bromo.) | tripartite motif containing 33 | TRIM33 |
| WDR9(2) | bromodomain and WD repeat domain containing 1, bromodomain 2 | BRWD1 |
Data Analysis & Interpretation
Definitions
Percent of Control (%Ctrl)
The results for single concentration (primary screen) binding interactions for tested compound(s) are reported in your study report and spreadsheets as '%Ctrl' and is calculated in the following manner:
test compound = client supplied compound
negative control = DMSO (100% control)
positive control = control compound (0% control)
Binding Constant (Kd)
The results for an 11-point dose response curve compound/kinase interactions are reported in your study report and spreadsheets as Kd, which are values derived using the Hill equation:
The Hill Slope is set to -1. Curves are fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.
Selectivity Score (S-Scores)
Selectivity Score or S-score is a quantitative measure of compound selectivity. It is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants.
This value can be calculated using %Ctrl as a potency threshold (below) and provides a quantitative method of describing compound selectivity to facilitate comparison of different compounds.
S(35) = (number of non-mutant kinases with %Ctrl <35)/(number of non-mutant kinases tested)
S(10) = (number of non-mutant kinases with %Ctrl <10)/(number of non-mutant kinases tested)
S(1) = (number of non-mutant kinases with %Ctrl <1)/(number of non-mutant kinases tested)
Using S-Score Data to Quantitate Selectivity
Selectivity Profile for 38 Small Molecule Kinase Inhibitors
KINOMEscan's in vitro competition binding assay was used to evaluate 38 kinase inhibitors against a panel of 287 distinct human protein kinases (~55% of the predicted human protein kinome), and three lipid kinases. The compounds tested included 21 tyrosine kinase inhibitors, 15 serine-threonine kinase inhibitors, 1 lipid kinase inhibitor, and staurosporine. S(35), 10uM = (number of non-mutant kinases with %Ctrl <35)/(290 kinases tested; 27 mutant variants were excluded from this analysis). Compounds approved for use in humans (as of August, 2007) are highlighted (gray bars) [click graph to enlarge].
TREEspot Kinase Dendrograms: A Visual Representation of Compound Selectivity
TREEspot is an innovative compound profile visualization tool for visualizing screening data. Kinases found to bind are marked with red circles, where larger circles indicate higher-affinity binding.
RSK Inhibitor
RSK inhibitor was reported in published findings to be a selective RSK kinase inhibitor on the basis of kinase profiling against a panel of 54 kinases. Selectivity profiling against the scanMAX kinase assay panel (then 402) revealed additional interactions not previously identified [click graph to enlarge].
TREEspot Compound Profile Visualization Tool
TREEspot is an innovative, secure access, web-based, compound profile visualization tool for analysis of KINOMEscan screening data. TREEspot is an ideal companion tool for existing data analysis applications and facilitates compound profile visualization through its simple yet powerful user interface.
Gain new perspectives of kinase profile data in a visual environment and distill essential knowledge to drive your discovery programs. TREEspot is provided as a complimentary tool to our clients. To learn more about how you can visualize your data using TREEspot or to request access credentials, please contact KINOMEscan.
- Easy-to-use
- Facilitates evaluation and analysis of profiling data
- Generates publication quality of TREEspot images
- Provides global visualization of profile data
- Visualize your data in a whole new way
