In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising ...approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610).
Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. Here, we ...demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC. Ectopic expression of either IRF4 or MYC antagonizes the phenotypic and transcriptional effects of CBP/EP300 bromodomain inhibition, highlighting the IRF4/MYC axis as a key component of its mechanism of action. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network.
The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ...ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure–activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 μM, BRET IC50 = 0.41 μM, BRD4(1) IC50 = 13 μM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.
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Bromodomain-containing protein 9 (BRD9), an epigenetic “reader” of acetylated lysines on post-translationally modified histone proteins, is upregulated in multiple cancer cell lines. ...To assess the functional role of BRD9 in cancer cell lines, we identified a small-molecule inhibitor of the BRD9 bromodomain. Starting from a pyrrolopyridone lead, we used structure-based drug design to identify a potent and highly selective in vitro tool compound 11, (GNE-375). While this compound showed minimal effects in cell viability or gene expression assays, it showed remarkable potency in preventing the emergence of a drug tolerant population in EGFR mutant PC9 cells treated with EGFR inhibitors. Such tolerance has been linked to an altered epigenetic state, and 11 decreased BRD9 binding to chromatin, and this was associated with decreased expression of ALDH1A1, a gene previously shown to be important in drug tolerance. BRD9 inhibitors may therefore show utility in preventing epigenetically-defined drug resistance.
CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential ...therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzob1,4diazepin-2-one.
The identification of a novel series of small molecule BET inhibitors is described. Using crystallographic binding modes of an amino-isoxazole fragment and known BET inhibitors, a structure-based ...drug design effort lead to a novel isoxazole azepine scaffold. This scaffold showed good potency in biochemical and cellular assays and oral activity in an in vivo model of BET inhibition.
Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma. eLife 5:e10483. doi: 10.7554/eLife.10483. Includes qPCR ...primer sequences and UPL probe numbers for RT-qPCR experiments described in the manuscript.
Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and ...inflammatory disease, respectively. Herein we report substituted benzobisoxazolo4,5-dazepines and benzotriazolo4,3-d1,4diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice.
The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of ...individual bromodomain proteins. A ligand-efficient nonselective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment. Small hydrophobic substituents replacing the N-methyl group were designed directing toward the conserved bromodomain water pocket, and two distinct binding conformations were then observed. The substituents either directly displaced and rearranged the conserved solvent network, as in BRD4(1) and TAF1(2), or induced a narrow hydrophobic channel adjacent to the lipophilic shelf, as in BRD9 and CECR2. The preference of distinct substituents for individual bromodomains provided selectivity handles useful for future lead optimization efforts for selective BRD9, CECR2, and TAF1(2) inhibitors.
Abstract
Triple negative breast cancer (TNBC) remains a high unmet medical need, commonly treated with chemotherapies. Biomarker-driven treatment strategies have led to the recent approvals of both ...PARP inhibitors and an immune checkpoint inhibitor for a subset of women with TNBC. Androgen receptor (AR) is expressed in a 20-30% of patients with TNBC. Preclinical studies indicate that AR in this context can substitute for estrogen receptor (ER) to drive transcription of ER target genes, supporting TNBC tumor proliferation and survival. AR-driven transcription is dependent upon multiple essential cofactors, among which are the structurally related, bromodomain (BRD) and lysine acetyl transferase containing proteins, CBP and p300. Here we report the discovery of FT-6876, a potent and selective small molecule bromodomain inhibitor of CBP/p300. Our hypothesis is that modulation of CBP/p300 with FT-6876 will alter chromatin compaction and, when in the vicinity of AR responsive genes, selectively reduce AR transcriptional activity at these loci. This should translate to growth inhibition in vitro and in vivo in AR+ breast cancer models. Consistent with this hypothesis, a rapid reduction in H3K27Ac was observed in an AR+ breast cancer cell line following treatment with FT-6876. Removal of the compound led to the restoration of this mark to baseline levels reflecting the dynamic state of histone modification in cells. High content analysis revealed that the reduction in H3K27Ac occurred in the majority of the cells. In order to have a greater understanding of the dynamic and selective impact of FT-6876 on AR/ER transcriptional activity, we used precision run-on (PRO) seq to quantify nascent RNA and infer transcription factor modulation genome-wide. GSEA analysis of nascent RNA showed a high enrichment for AR and ER pathway genes upon exposure with FT-6876. Using ChIPseq, we confirmed that the reduction of H3K27Ac at specific promoter sites was concurrent with a decrease in CBP/p300 on the chromatin and reduction in nascent RNA and eRNA. FT-6876 reduced proliferation in a time-dependent manner with optimal growth inhibition observed after 10 days drug exposure. This was maintained even after removal of the drug for several days. Across a panel of breast cancer cell lines, there was a good correlation between growth inhibition and AR expression. In vivo, FT-6876 induced tumor stasis at well tolerated doses in AR+ breast cancer models. This was associated with a reduction in H3K27Ac, AR and ER target gene modulation, and reduction in Ki67. FT-6876 is a promising new CBP/p300 bromodomain inhibitor demonstrating efficacy in preclinical models of AR+ breast cancer.
Citation Format: Maureen Caligiuri, Grace L. Williams, Jennifer Castro, Linda Battalagine, Kristina Muskiewicz, Erik Wilker, Lili Yao, Shawn Schiller, Angela Toms, Ping Li, Eneida Pardo, Bradford Graves, Kenneth W. Wood, Sylvie M. Guichard. FT-6876, a potent and selective inhibitor of CBP/p300 with antitumor activity in AR-positive breast cancer abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3079.
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