Based on these data, we have concluded that these compounds, at least in our hands, are interfering with the TRIM24 and TRIM33 AlphaScreen assay, and do not bind to TRIM33β. However, the use of different protein isoforms my account for some of the observed discrepancies. Our data show that all five compounds interfere with our TRIM24 and TRIM33 AlphaScreen assays. We could not detect binding of compounds 3 or 4 to the TRIM33β BRD using either ITC or waterLOGSY. To select compounds to be used in the AlphaScreen assay, 31743 compounds from the Maybridge fragment library (Fisher Scientific), along with 1534 compounds from the PPI-Net collection, were screened in silico against TRIM24 and TRIM33α. Based on the results from this screen, we identified an enriched fragment library of 200 compounds selected for predicted affinity, reliability of binding mode, and synthetic tractability. These compounds, along with the entire PPI-Net library, were screened against TRIM24, TRIM33α and TRIM33β using a qualitative AlphaScreen response assay (data not shown). In this assay, the compounds were tested at two concentrations, 30 μM and 150 μM, as a rapid method of establishing activity. This study identified seven molecules from the PPI-Net library, and four molecules from the Maybridge library, as potential binders to one or more of the TRIM24 and/or TRIM33 reader domains. To increase the number of putative ligands we identified additional ligands in the Maybridge Fragment library that show similarity to the hits, based on the Tanimoto coefficient. We only selected ligands that have a solubility forecast index (SFI) 62 of <6.5. After validation of these molecules using NMR WaterLOGSY experiments and AlphaScreen TruHits screens (data not shown), four promising lead compounds 8-11 were identified (Figure ). TRIM33β N1039F mutant, as would be expected for a compound binding to the BRD. The binding of this compound was unaffected by the W889A or W828A mutants, indicating that it does not bind to the PHD of either TRIM24 or TRIM33. Compound 9 showed no binding to TRIM24, but it does bind to the WT TRIM33α and TRIM33β. Binding to TRIM33β, but not TRIM33α, was disrupted by both the W889A and N1039F mutations, suggesting that this compound interacts with both the BRD and PHD or binds in between these domains. Compounds 10 and 11 bind to all three proteins, and their binding was unaffected by any of the mutants, indicating that these compounds do not bind to the BRD or PHD.