Only two high affinity probes have been reported for the TRIM24 BRD, and both compounds also bind to the BRPF1 BRD (Figure ). A patent from Qi and Pei claims for a series of TRIM33 BRD ligands identified using an AlphaScreen (Figure ). The isoform of TRIM33 used in this work is not specified in the patent, but was later confirmed to be TRIM33α (Qi personal communication). We investigated using the previously reported TRIM24 and TRIM33 ligands to help validate our assay. Our AlphaScreen assay results for the TRIM24 ligand 2 agree with the literature reports (Figure ), as displacement of H3 1-27 K18Ac was observed for TRIM24, but not for either isoform of TRIM33. A K i value of 219 nM was obtained for compound 2 binding to the TRIM24 BRD-PHD, which is very similar to the K d value of 222 nM reported by Bennett et al. Three of the reported TRIM33 ligands (3, 4 and 6) showed apparent displacement of all three peptides from all three proteins in the AlphaScreen assay, with the additional two showing binding to TRIM24 and either weak or no binding to TRIM33 (Table , compounds 3-7). However, an AlphaScreen TruHits screen 60 indicated significant assay interference from all five compounds (3-7, Table ), meaning that AlphaScreen data generated for these compounds are unreliable. We therefore sought to investigate TRIM33 binding of compounds 3 and 4 using ITC and waterLOGSY approaches [Figures ]. These compounds were selected as they have a range of reported IC 50 values for TRIM33. Neither compound showed binding to TRIM33β when assessed by ITC (Figure -4), using conditions based on the reported IC 50 values. To determine whether these compounds are weak binders to TRIM33β we subjected them to a waterLOGSY assay against TRIM33β. Again, neither compound showed any binding to this protein (Figures ).