Development of a Fluorescence Polarization Assay for p300/CBP and Its Application Using a Direct-to-Biology Approach.

The histone acetyltransferases E1A binding protein of 300 kDa (p300) and its homologue cyclic AMP response element binding protein (CREB) binding protein (CBP) are potential targets for cancer treatment. However, no drugs targeting p300/CBP have yet been approved. Various bioassay methods have been developed to evaluate the inhibitory potency of the corresponding inhibitors. However, suitable assays for high-throughput screening (HTS) of novel inhibitors targeting the p300/CBP bromodomain are lacking. To address this shortcoming, we developed a fluorescence polarization (FP) assay. This employs a rationally designed strategy that exhibits excellent characteristics, making it suitable for the large-scale evaluation of compound bioactivity. To enable direct-to-biology application of this FP assay, we constructed an 840-compound library on microplates via the CuAAC reaction and performed HTS. Using this platform, we rapidly identified a series of small molecules that target the p300 bromodomain and exhibit potent activity. Computational chemistry studies have provided insight into the binding mode of the inhibitors, while cellular studies and H3K27 acetylation levels demonstrate the favorable properties of these compounds against acute myeloid leukemia (AML). Overall, this platform demonstrates a multifunctional approach, integrating rational FP probe design with combinatorial chemistry. This closes a methodological gap in the rapid discovery of p300/CBP bromodomain inhibitors, providing a new paradigm for accelerating drug discovery.