A new BET on the control of HIV latency

The mammalian target of rapamycin (mTOR) signaling pathway integrates signals from the environment to the nucleus for the regulation of cellular growth, metabolism and survival. Lymphocytes frequently rely on this pathway, but it is carefully regulated through the reception of signals via cytokine, growth factor, and co-stimulatory receptors. Recent studies have begun to elucidate why T cell subsets rely on this pathway to varying degrees. Ultimately these findings will help distinguish the parameters that guide T cell homeostasis and activation-induced function between the different T cell populations. The mTOR pathway has been the focus of many immunosuppressive and cancer treatment regimens, therefore there is a great need to understand the impact of suppression not only on the T cell populations targeted, but on bystander T cells as well.     

Lead optimization and efficacy evaluation of quinazoline-based BET family inhibitors for potential treatment of cancer and inflammatory diseases

Extensive optimization of quinazoline-based lead 8 is described. The structure-activity relationship studies indicate the S-configuration is preferred for the phenylmorpholine substitution. Together with incorporation of a (2-hydroxyl-2-methylpropyl)pyrazole moiety at the 2-position leads to analogs with comparable potency and marked improvement in the pharmacokinetic profile over our previously reported lead compounds. Further in vivo efficacy studies in Kasumi-1 xenograft mouse model demonstrates that the selected inhibitors are well tolerated and highly efficacious in the inhibition of tumor growth. Additionally, the representative analog 19 also demonstrated significant improvement of arthritis severity in a collagen-induced arthritis (CIA) mouse model. These results indicate potential use of these quinazoline-based BET inhibitors for treatment of cancer and inflammatory diseases. A brief discussion of the co-crystallized structure of 19 with BRD4 (BD1) is also highlighted.     

Methylpyrrole inhibitors of BET bromodomains

An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma.     

Co-targeting BET bromodomain BRD4 and RAC1 suppresses growth,stemness and tumorigenesis by disrupting the c-MYC-G9a-FTH1axis and downregulating HDAC1 in molecular subtypes of breast cancer

BET bromodomain BRD4 and RAC1 oncogenes are considered important therapeutic targets for cancer and play key roles in tumorigenesis, survival and metastasis. However, combined inhibition of BRD4-RAC1 signaling pathways in different molecular subtypes of breast cancer including luminal-A, HER-2 positive and triple-negative breast (TNBC) largely remains unknown. Here, we demonstrated a new co-targeting strategy by combined inhibition of BRD4-RAC1 oncogenic signaling in different molecular subtypes of breast cancer in a context-dependent manner. We show that combined treatment of JQ1 (inhibitor of BRD4) and NSC23766 (inhibitor of RAC1) suppresses cell growth, clonogenic potential, cell migration and mammary stem cells expansion and induces autophagy and cellular senescence in molecular subtypes of breast cancer cells. Mechanistically, JQ1/NSC23766 combined treatment disrupts MYC/G9a axis and subsequently enhances FTH1 to exert antitumor effects. Furthermore, combined treatment targets HDAC1/Ac-H3K9 axis, thus suggesting a role of this combination in histone modification and chromatin modeling. C-MYC depletion and co-treatment with vitamin-C sensitizes different molecular subtypes of breast cancer cells to JQ1/NSC23766 combination and further reduces cell growth, cell migration and mammosphere formation. Importantly, co-targeting RAC1-BRD4 suppresses breast tumor growth in vivo using xenograft mouse model. Clinically, RAC1 and BRD4 expression positively correlates in breast cancer patient''s samples and show high expression patterns across different molecular subtypes of breast cancer. Both RAC1 and BRD4 proteins predict poor survival in breast cancer patients. Taken together, our results suggest that combined inhibition of BRD4-RAC1 pathways represents a novel and potential therapeutic approach in different molecular subtypes of breast cancer and highlights the importance of co-targeting RAC1-BRD4 signaling in breast tumorigenesis via disruption of C-MYC/G9a/FTH1 axis and down regulation of HDAC1.     

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.     

Discovery of novel [1,2,4]triazolo[4,3-a]quinoxaline aminophenyl derivatives as BET inhibitors for cancer treatment

Bromodomain and extra-terminal (BET) proteins, a class of epigenetic reader domains has emerged as a promising new target class for small molecule drug discovery for the treatment of cancer, inflammatory, and autoimmune diseases. Starting from in silico screening campaign, herein we report the discovery of novel BET inhibitors based on [1,2,4]triazolo[4,3-a]quinoxaline scaffold and their biological evaluation. The hit compound was optimized using the medicinal chemistry approach to the lead compound with excellent inhibitory activities against BRD4 in the binding assay. The substantial antiproliferative activities in human cancer cell lines, promising drug-like properties, and the selectivity for the BET family make the lead compound (13) as a novel BRD4 inhibitor motif for anti-cancer drug discovery.     

Inhibition of bromodomain and extra‐terminal proteins increases sensitivity to venetoclax in chronic lymphocytic leukaemia

The development of drugs able to target BTK, PI3k‐delta and BCL2 has dramatically improved chronic lymphocytic leukaemia (CLL) therapies. However, drug resistance to these therapies has already been reported due to non‐recurrent changes in oncogenic pathways and genes expression signatures. In this study, we investigated the cooperative role of the BCL2 inhibitor venetoclax and the BRD4 inhibitor JQ1. In particular, we found that JQ1 shows additional activity with venetoclax, in CLL cell lines and in ex vivo isolated primary CD19⁺ lymphocytes, arguing in favour of combination strategies. Lastly, JQ1 is also effective in venetoclax‐resistant CLL cell lines. Together, our findings indicated that the BET inhibitor JQ1 could be a promising therapy in CLL, both as first‐line therapy in combination with venetoclax and as second‐line therapy, after the emergence of venetoclax‐resistant clones.     

Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains

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Exploiting a water network to achieve enthalpy-driven,bromodomain-selective BET inhibitors

Within the last decade, the Bromodomain and Extra-Terminal domain family (BET) of proteins have emerged as promising drug targets in diverse clinical indications including oncology, auto-immune disease, heart failure, and male contraception. The BET family consists of four isoforms (BRD2, BRD3, BRD4, and BRDT/BRDT6) which are distinguished by the presence of two tandem bromodomains (BD1 and BD2) that independently recognize acetylated-lysine (KAc) residues and appear to have distinct biological roles. BET BD1 and BD2 bromodomains differ at five positions near the substrate binding pocket: the variation in the ZA channel induces different water networks nearby. We designed a set of congeneric 2- and 3-heteroaryl substituted tetrahydroquinolines (THQ) to differentially engage bound waters in the ZA channel with the goal of achieving bromodomain selectivity. SJ830599 (9) showed modest, but consistent, selectivity for BRD2-BD2. Using isothermal titration calorimetry, we showed that the binding of all THQ analogs in our study to either of the two bromodomains was enthalpy driven. Remarkably, the binding of 9 to BRD2-BD2 was marked by negative entropy and was entirely driven by enthalpy, consistent with significant restriction of conformational flexibility and/or engagement with bound waters. Co-crystallography studies confirmed that 9 did indeed stabilize a water-mediated hydrogen bond network. Finally, we report that 9 retained cytotoxicity against several pediatric cancer cell lines with EC₅₀ values comparable to BET inhibitor (BETi) clinical candidates.     

Domain-selective targeting of BET proteins in cancer and immunological diseases

Cancer and inflammation are strongly interconnected processes. Chronic inflammatory pathologies can be at the heart of tumor development; similarly, tumor-elicited inflammation is a consequence of many cancers. The mechanistic interdependence between cancer and inflammatory pathologies points toward common protein effectors which represent potential shared targets for pharmacological intervention. Epigenetic mechanisms often drive resistance to cancer therapy and immunomodulatory strategies. The bromodomain and extraterminal domain (BET) proteins are epigenetic adapters which play a major role in controlling cell proliferation and the production of inflammatory mediators. A plethora of small molecules aimed at inhibiting BET protein function to treat cancer and inflammatory diseases have populated academic and industry efforts in the last 10 years. In this review, we will discuss recent pharmacological approaches aimed at targeting a single or a subset of the eight bromodomains within the BET family which have the potential to tease apart clinical efficacy and safety signals of BET inhibitors.