Discovery and lead identification of quinazoline-based BRD4 inhibitors

A new series of quinazoline-based analogs as potent bromodomain-containing protein 4 (BRD4) inhibitors is described. The structure-activity relationships on 2- and 4-position of quinazoline ring, and the substitution at 6-position that mimic the acetylated lysine are discussed. A co-crystallized structure of 48 (CN750) with BRD4 (BD1) including key inhibitor-protein interactions is also highlighted. Together with preliminary rodent pharmacokinetic results, a new lead (65, CN427) is identified which is suitable for further lead optimization.     

Protein targeting chimeric molecules specific for dual bromodomain 4 (BRD4) and Polo-like kinase 1 (PLK1) proteins in acute myeloid leukemia cells

Proteolysis targeting chimeras (PROTACs) are hetero-bifunctional molecules that could simultaneously bind to the target protein and the E3 ubiquitin ligase, thereby leading to selective degradation of the target protein. Polo-like kinase 1 (PLK1) and bromodomain 4 (BRD4) are both attractive therapeutic targets in acute myeloid leukemia (AML). Here, we developed a small-molecule BRD4 and PLK1 degrader HBL-4 based on PROTAC technology, which leads to fast, efficient, and prolonged degradation of BRD4 and PLK1 in MV4-11 cells tested in vitro and vivo, and potent anti-proliferation and BRD4 and PLK1 degradation ability in human acute leukemia MOLM-13 and KG1 cells. Meanwhile, HBL-4 more effectively suppresses c-Myc levels than inhibitor BI2536, resulting in more effective inducing apoptosis activity in MV4-11 cells. At the same time, HBL-4 induced dramatically improved efficacy in the MV4-11 tumor xenograft model as compared with BI2536. This study is, to our knowledge, the first reports about dual PLK1 and BRD4 degraders, which potentially represents an important therapeutic advance in the treatment of cancer.     

Genomic profiling of solid tumors harboring BRD4-NUT and response to immune checkpoint inhibitors

BackgroundThe translocation t(15:19) produces the oncogenic BRD4-NUT fusion which is pathognomonic for NUT carcinoma (NC), which is a rare, but extremely aggressive solid tumor. Comprehensive genomic profiling (CGP) by hybrid-capture based next generation sequencing of 186+ genes of a cohort of advanced cancer cases with a variety of initial diagnoses harboring BRD4-NUT may shed further insight into the biology of these tumors and possible options for targeted treatment.Case presentationThirty-one solid tumor cases harboring a BRD4-NUT translocation are described, with only 16% initially diagnosed as NC and the remainder carrying other diagnoses, most commonly NSCLC—NOS (22%) and lung squamous cell carcinoma (NSCLC-SCC) (16%). The cohort was all microsatellite stable and harbored a low Tumor Mutational Burden (TMB, mean 1.7 mut/mb, range 0–4). In two index cases, patients treated with immune checkpoint inhibitors (ICPI) had unexpected partial or better responses of varying duration. Notably, four cases – including the two index cases - were negative for PD-L1 expression. Neo-antigen prediction for BRD4-NUT and then affinity modeling of the peptide-MHC (pMHC) complex for an assessable index case predicted very high affinity binding, both on a ranked (99.9%) and absolute (33 nM) basis.ConclusionsCGP identifies BRD4-NUT fusions in advanced solid tumors which carry a broad range of initial diagnoses and which should be re-diagnosed as NC per guidelines. A hypothesized mechanism underlying responses to ICPI in the low TMB, PD-L1 negative index cases is the predicted high affinity of the BRD4-NUT fusion peptide to MHC complexes. Further study of pMHC affinity and response to immune checkpoint inhibitors in patients with NC harboring BRD4-NUT is needed to validate this therapeutic hypothesis.     

BRD4在喉鳞状细胞癌病灶转移过程中的作用及机制研究

目的:探讨溴样结构域蛋白4 (Bromoid Domain Protein 4,BRD4)在喉鳞状细胞癌病灶转移过程中的作用及其机制。方法:收集本院2016年4月-2018年4月收治的87例喉鳞状细胞癌患者手术标本。qRT-PCR、Western blot和免疫组化染色检测患者肿瘤组织(Tumor Tissue)、癌旁组织(Adjacent Tissue)和正常组织(Normal Tissue)中BRD4表达;BRD4拮抗剂GSK525762A(500nmo L/L)处理喉鳞状细胞癌Hep2细胞,24、48和72 h后CCK-8检测细胞增殖;Trans-well细胞迁移实验和侵袭实验分别检测GSK525762A给药前后Hep2细胞迁移和侵袭能力;qRT-PCR和Western blot检测GSK525762A给药前后Hep2细胞BRD4、E-Cadherin、N-Cadherin和Twist蛋白表达。结果:喉鳞状细胞癌肿瘤组织中BRD4表达高于癌旁组织和正常卵巢组织(P0.05);GSK525762A给药处理后Hep2细胞增殖能力、迁移和侵袭能力及BRD4和上皮间质转化相关蛋白N-Cadherin和Twist表达均明显低于阴性对照组(P0.05),而E-Cadherin表达的表达明显升高(P0.05)。结论:BRD4可能通过抑制E-Cadherin的表达,增加N-Cadherin和Twist的表达,进而激活上皮间质转化,促进喉鳞状细胞癌转移。     

Expression and clinical value of PD-L1 which is regulated by BRD4 in tongue squamous cell carcinoma

The programmed cell death-ligand-1 (PD-L1) and bromodomain protein 4 (BRD4) are frequently overexpressed in cancer and have even been shown to act synergistically. The aim of this study was to determine their potential oncogenic role .in tongue squamous cell carcinoma (TSCC). We detected significantly higher expression levels of both PD-L1 and BRD4 in TSCC tissues compared to normal tissues (P ≤ .05). In addition, the high levels of PD-L1 were significantly associated with increased tumor lymphatic metastasis (P ≤ .05), tumor staging (P ≤ .01), as well as BRD4 expression (P ≤ .05). Genetic and pharmacological inhibition of BRD4 in TSCC cells not only reduced their growth rate but also PD-L1 levels (P ≤ .05), while overexpression of BRD4 upregulated PD-L1. Bioinformatics analysis showed that c-MYC and CDK9 were interactive partners of both BRD4 and PD-L1. While c-MYC clearly modulated the expression of PD-L1, as well as reversed the inhibitory effects of JQ1, no obvious association was observed between CDK9 and PD-L1. We report a novel regulatory axis consisting of BRD4, PD-L1, and c-MYC that likely drives TSCC progression, and is a potential prognostic marker and/or therapeutic target for TSCC.     

Circular RNA circBCBM1 promotes breast cancer brain metastasis by modulating miR-125a/BRD4 axis

Circular RNAs (circRNAs) play critical roles in tumorigenesis and the progression of various cancers. We previously identified a novel upregulated circRNA, circBCBM1 (hsa_circ_0001944), in the context of breast cancer brain metastasis. However, the potential biological function and molecular mechanism of circBCBM1 in breast cancer brain metastasis remain largely unknown. In this study, we confirmed that circBCBM1 was a stable and cytoplasmic circRNA. Functionally, circBCBM1 promoted the proliferation and migration of 231-BR cells in vitro and growth and brain metastasis in vivo. Mechanistically, circBCBM1 acted as an endogenous miR-125a sponge to inhibit miR-125a activity, resulting in the upregulation of BRD4 (bromodomain containing 4) and subsequent upregulation of MMP9 (matrix metallopeptidase 9) through Sonic hedgehog (SHH) signaling pathway. Importantly, circBCBM1 was markedly upregulated in the breast cancer brain metastasis cells and clinical tissue and plasma samples; besides, circBCBM1 overexpression in primary cancerous tissues was associated with shorter brain metastasis-free survival (BMFS) of breast cancer patients. These findings indicate that circBCBM1 is involved in breast cancer brain metastasis via circBCBM1/miR-125a/BRD4 axis. CircBCBM1 may serve as a novel diagnostic and prognostic biomarker and potential therapeutic target for breast cancer brain metastasis.     

Discovery and biological evaluation of proteolysis targeting chimeras (PROTACs) as an EGFR degraders based on osimertinib and lenalidomide

Epidermal growth factor receptor (EGFR) is one of the important and valuable drug targets. Overexpression of EGFR is associated with the development of many types of cancer. In this study, three PROTACs small molecules (16a–16c) were designed, synthesized and evaluated for their cytotoxicity against the growth in different NSCLC cell line and the degradation effect. The bioassay results indicated that 16c has a good inhibition in PC9 cells and H1975 cells, and the corresponding IC₅₀ value was 0.413 μM and 0.657 μM, respectively. Western blotting results demonstrated that compound 16c could serve as an effective EGFR^del19-targeting degrader in PC9 cells.     

Design,synthesis and biological evaluation of novel indole derivatives as potential HDAC/BRD4 dual inhibitors and anti-leukemia agents

HDAC inhibitors and BRD4 inhibitors were considered to be potent anti-cancer agents. Recent studies have demonstrated that HDAC and BRD4 participate in the regulation of some signal paths like PI3K-AKT. In this work, a series of indole derivatives that combine the inhibitory activities of BRD4 and HDAC into one molecule were designed and synthesized through the structure-based design method. Most compounds showed potent HDAC inhibitory activity and moderate BRD4 inhibitory activity. In vitro anti-proliferation activities of the synthesized compounds were also evaluated. Among them, 19f was the most potent inhibitor against HDAC3 with IC₅₀ value of 5 nM and BRD4 inhibition rate of 88% at 10 μM. It was confirmed that 19f could up-regulate the expression of Ac-H3 and reduce the expression of c-Myc by western blot analysis. These results indicated that 19f was a potent dual HDAC/BRD4 inhibitor and deserved further investigation.     

Design,synthesis and biological evaluation of hypolipidemic compounds based on BRD4 inhibitor RVX-208

Bromodomain-containing protein 4 (BRD4) is a new therapeutic target for the treatment of diseases including cardiovascular diseases, cancer, inflammation and central nervous system (CNS) disorders. In this study, we introduced the pharmacophore of fibrates to a BRD4 inhibitor, RVX-208, to design dual-active hypolipidemic compounds, and found that some of new analogues showed favorable hypolipidemic activities. Synthetic accessibility towards this class of compounds optimized RVX-208 as well as would supply more thoughts on hypolipidemic drugs.     

BRD4 Promotes DNA Repair and Mediates the Formation of TMPRSS2-ERG Gene Rearrangements in Prostate Cancer

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Binding pocket-based design,synthesis and biological evaluation of novel selective BRD4-BD1 inhibitors

Bromodomain-containing protein 4 (BRD4), consisting of two tandem bromodomains (BD1 and BD2), is key epigenetic regulator in fibrosis and cancer, which has been reported that BD1 and BD2 have distinct roles in post-translational modification. But there are few selective inhibitors toward those two domains. Herein, this study designed and synthesized a series of novel selective BRD4-BD1 inhibitors, using computer-aided drug design (CADD) approach focused on exploring the difference of the binding pockets of BD1 and BD2, and finding the His437 a crucial way to achieve BRD4-BD1 selectivity. Our results revealed that the compound 3u is a potent selective BRD4-BD1 inhibitor with IC₅₀ values of 0.56?μM for BD1 but >100?μM for BD2. The compound exhibited a broad spectrum of anti-proliferative activity against several human cancer and fibroblastic cell lines, which might be related to its capability of reducing the expression of c-Myc and collagen I. Furthermore, it could induce apoptosis in A375 cells. To the contrary, the selective BD2 inhibitor, RVX-208, did not indicate any of these activities. Our findings highlight that the function of BRD4-BD1 might be predominant in fibrosis and cancer. And it is rational to further develop novel selective BRD4-BD1 inhibitors.     

Design,synthesis and biological evaluation of novel 6-phenyl-1,3a,4,10b-tetrahydro-2H-benzo[c]thiazolo[4,5-e]azepin-2-one derivatives as potential BRD4 inhibitors

Bromodomain-containing protein 4 (BRD4) is a key epigenetic regulator in cancer, and inhibitors targeting BRD4 exhibit great anticancer activity. By replacing the methyltriazole ring of the BRD4 inhibitor I-BET-762 with an N-methylthiazolidone heterocyclic ring, fifteen novel BRD4 inhibitors were designed and synthesized. Compound 13f had a hydrophobic acetylcyclopentanyl side chain, showing the most potent BRD4 inhibitory activity in the BRD4-BD1 inhibition assay (IC₅₀ value of 110 nM), it also significantly suppressed the proliferation of MV-4-11 cells with high BRD4 level (IC₅₀ value of 0.42 μM). Furthermore, the potent apoptosis-promoting and G0/G1 cycle-arresting activity of compound 13f were indicated by flow cytometry. As the downstream-protein of BRD4, c-Myc was in significantly low expression by compound 13f treatment in a dose-dependent manner. All the findings supported that this novel compound 13f provided a perspective for developing effective BRD4 inhibitors.