| In Vitro | In vitro activity: CeMMEC1 only bound BRD4 very weakly but it shows high affinity for the bromodomains of CREBBP, EP300, BRD9 and the second bromodomain of TAF1.
Cell Assay: Bromodomain-containing proteins of the BET family recognize histone lysine acetylation and mediate transcriptional activation of target genes such as the MYC oncogene. Pharmacological inhibitors of BET domains promise therapeutic benefits in a variety of cancers. We performed a high-diversity chemical compound screen for agents capable of modulating BRD4-dependent heterochromatization of a generic reporter in human cells. In addition to known and new compounds targeting BRD4, we identified small molecules that mimic BRD4 inhibition without direct engagement. One such compound was a potent inhibitor of the second bromodomain of TAF1. Using this inhibitor, we discovered that TAF1 synergizes with BRD4 to control proliferation of cancer cells, making TAF1 an attractive epigenetic target in cancers driven by MYC. |
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| Kinase Assay | TAF1 binding assays are conducted using the EPIgeneous Binding Domain kit B. Binding is determined by the displacement of an acetylated biotin peptide from a GST-tagged TAF1 protein using HTRF with a Eu3+-conjugated GST antibody donor and streptavidin-conjugated acceptor. Compounds (CeMMEC1) are dispensed into assay plates, ProxiPlate-384 Plus using an Echo 525 Liquid Handler. Binding assays are conducted in a final volume of 20 μL with 5 nM TAF1-GST, 50 nM peptide (SGRGK (ac)GGK (ac)GLGK (ac)GGAK (ac)RHRK (biotin)-acid), 6.25 nM Streptavidin-XL665, 1:200 Anti-GST-Eu3+ cryptate and 0.1% DMSO. Assay reagents are dispensed into plates using a Multidrop combi and incubated at room temperature for 3 h. Fluorescence is measured using a PHERAstar microplate reader using the HTRF module with dual emission protocol (A = excitation of 320 nm, emmission of 665 nm, and B = excitation of 320 nm, emission of 620 nm). Raw data are processed to give an HTRF ratio (channel A/B × 10,000), which is used to generate IC50 curves. |
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