Discovery of Dual TAF1-ATR Inhibitors and Ligand-Induced Structural Changes of the TAF1 Tandem Bromodomain.

Bromodomains regulate chromatin remodeling and gene transcription through recognition of acetylated lysines on histones and other proteins. Bromodomain-containing protein TAF1, a subunit of general transcription factor TFIID, initiates preinitiation complex formation and cellular transcription. TAF1 serves as a cofactor for certain oncogenic transcription factors and is implicated in regulating the p53 tumor suppressor. Therefore, TAF1 is a potential target to develop small molecule therapeutics for diseases arising from dysregulated transcription, such as cancer. Here, we report the ATR kinase inhibitor AZD6738 (Ceralasertib) and analogues thereof as bona fide inhibitors of TAF1. Crystallographic and small-angle X-ray scattering studies established that newly identified and previously reported inhibitors stabilize distinct structural states of the TAF1 tandem bromodomain through "open-closed" transitions and dimerization. Combined with functional studies on p53 signaling in cancer cell lines, the data provide new insights into the feasibility and challenges of TAF1 inhibitors as chemical probes and therapeutics.

EP300 Selectively Controls the Enhancer Landscape of MYCN-Amplified Neuroblastoma.

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. EP300 controls enhancer acetylation by interacting with TFAP2ß, a transcription factor member of the lineage-defining transcriptional core regulatory circuitry (CRC) in NB. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed "JQAD1" that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at CRC enhancers and rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. Furthermore, JQAD1 activity is critically determined by cereblon (CRBN) expression across NB cells. SIGNIFICANCE: EP300, but not CBP, controls oncogenic CRC-driven transcription in high-risk NB by binding TFAP2ß. We developed JQAD1, a CRBN-dependent PROTAC degrader with preferential activity against EP300 and demonstrated its activity in NB. JQAD1 has limited toxicity to untransformed cells and is effective in vivo in a CRBN-dependent manner. This article is highlighted in the In This Issue feature, p. 587.

Development of Dimethylisoxazole-Attached Imidazo[1,2-a]pyridines as Potent and Selective CBP/P300 Inhibitors.

The use of epigenetic bromodomain inhibitors as anticancer therapeutics has transitioned from targeting bromodomain extraterminal domain (BET) proteins into targeting non-BET bromodomains. The two most relevant non-BET bromodomain oncology targets are cyclic AMP response element-binding protein (CBP) and E1A binding protein P300 (EP300). To explore the growing CBP/EP300 interest, we developed a highly efficient two-step synthetic route for dimethylisoxazole-attached imidazo[1,2-a]pyridine scaffold-containing inhibitors. Our efficient two-step reactions enabled high-throughput synthesis of compounds designed by molecular modeling, which together with structure-activity relationship (SAR) studies facilitated an overarching understanding of selective targeting of CBP/EP300 over non-BET bromodomains. This led to the identification of a new potent and selective CBP/EP300 bromodomain inhibitor, UMB298 (compound 23, CBP IC50 72 nM and bromodomain 4, BRD4 IC50 5193 nM). The SAR we established is in good agreement with literature-reported CBP inhibitors, such as CBP30, and demonstrates the advantage of utilizing our two-step approach for inhibitor development of other bromodomains.