Discovery Of Inhibitors Targeting The Bromodomain Of The Histone Acetylation Recognition Factor BPTF

The Bromodomain（BRD）family of proteins is a recognition factor for histone lysine acetylation and can specifically bind to it.BPTF（Bromodomain PHD finger transcription factor）is a member of the BRD family,which can recognize acetylated histone lysine sites and play an important role in chromatin remodeling,transcription regulation and embryonic development.Mutation and dislocation of BPTF gene,as well as dysregulation of expression,have an important impact on its function,especially in the occurrence and development of many cancers.In the first part of this study,we firstly analyzed and investigated the existing BPTF-BRD inhibitors,and selected the small molecule inhibitor TP-238 with high activity to fragment it to obtain its potential active group fragment.Furthermore,it was structurally modified by chemical synthesis to obtain the hit compound DC-BPi-03,and its activity IC₅₀ value was 698.3±21.0 n M.Then,through the analysis of the complex crystal structure of the hit compound and BPTF-BRD,the binding mode was further clarified and the specific medicinal chemistry optimization direction was provided,so that we obtained a BPTF-BRD inhibitor with higher affinity and selectivity DC-BPi-07(IC₅₀=16.0±5.0 n M).Subsequently,by analyzing the co-crystal structure of the complex of BPTF-BRD and DC-BPi-07,the reasons for the increased activity and selectivity were verified,which provided a chemical probe for the subsequent exploration of its mode of action at the cellular level.Further cell-level studies showed that DC-BPi-07 could inhibit the proliferation of human myelomonocytic leukemia cell MV-4-11 in a concentration-dependent manner,and its inhibitory activity was 5.2μM.Finally,we demonstrated the regulation of DC-BPi-07on the expression of BPTF downstream target genes c-MYC,BCL2 and CDK6 by RT-q PCR.In order to discover BPTF-BRD inhibitors with a new backbone,in the second part of the work,we screened the inhibitors against the laboratory-built compound library,and obtained the small molecule inhibitor Sanguinarine Chloride.Its molecular level inhibitory activity IC₅₀ value is 344.2±21.5 n M.Further,we confirmed the direct binding of the compound to BPTF-BRD by surface plasmon resonance experiments with an affinity K_D of 287.3 n M.We simulated and analyzed the binding mode of Sanguinarine Chloride to BPTF-BRD by molecular docking experiment.Subsequently,we determined the proliferation inhibitory effect of sanguinarine on MV-4-11,and its activity was 0.7μM,indicating that Sanguinarine Chloride has a certain antitumor effect.Finally,we preliminarily verified that Sanguinarine Chloride can inhibit the proliferation of leukemia cells by regulating the expression of the downstream cancer gene c-MYC by western blotting.In conclusion,based on our study,DC-BPi-07 has the potential as a chemical probe to study how BPTF is involved in c-MYC transcription-related cancers,and can also serve as a starting point for the development of clinical drug candidates.And Sanguinarine Chloride can provide a new chemical framework for the research of bromodomain family inhibitors.