BRD4 Research Articles

Dopamine Transporter Knockout Rats Display Epigenetic Alterations in Response to Cocaine Exposure.

(1) Background: There is an urgent need for effective treatments for cocaine use disorder (CUD), and new pharmacological approaches targeting epigenetic mechanisms appear to be promising options for the treatment of this disease. Dopamine Transporter (DAT) transgenic rats recently have been proposed as a new animal model for studying susceptibility to CUD. (2) Methods: DAT transgenic rats were treated chronically with cocaine (10 mg/kg) for 8 days, and the expression of epigenetic modulators, Lysine Demethylase 6B (KDM6B) and Bromodomain-containing protein 4 (BRD4), was examined in the prefrontal cortex (PFC). (3) Results: We show that only full knockout (KO) of DAT impacts basal levels of KDM6B in females. Additionally, cocaine altered the expression of both epigenetic markers in a sex- and genotype-dependent manner. In response to chronic cocaine, KDM6B expression was decreased in male rats with partial DAT mutation (HET), while no changes were observed in wild-type (WT) or KO rats. Indeed, while HET male rats have reduced KDM6B and BRD4 expression, HET female rats showed increased KDM6B and BRD4 expression levels, highlighting the impact of sex on epigenetic mechanisms in response to cocaine. Finally, both male and female KO rats showed increased expression of BRD4, but only KO females exhibited significantly increased KDM6B expression in response to cocaine. Additionally, the magnitude of these effects was bigger in females when compared to males for both epigenetic enzymes. (4) Conclusions: This preliminary study provides additional support that targeting KDM6B and/or BRD4 may potentially be therapeutic in treating addiction-related behaviors in a sex-dependent manner.

BRD7 improves glucose homeostasis independent of IRS proteins.

Bromodomain-containing protein 7 (BRD7) has emerged as a player in the regulation of glucose homeostasis. Hepatic BRD7 levels are decreased in obese mice, and the reinstatement of hepatic BRD7 in obese mice has been shown to establish euglycemia and improve glucose homeostasis. Of note, the upregulation of hepatic BRD7 levels activates the AKT cascade in response to insulin without enhancing the sensitivity of the insulin receptor (InsR)-insulin receptor substrate (IRS) axis. In this report, we provide evidence for the existence of an alternative insulin signaling pathway that operates independently of IRS proteins and demonstrate the involvement of BRD7 in this pathway. To investigate the involvement of BRD7 as a downstream component of InsR, we utilized liver-specific InsR knockout mice. Additionally, we employed liver-specific IRS1/2 knockout mice to examine the requirement of IRS1/2 for the action of BRD7. Our investigation of glucose metabolism parameters and insulin signaling unveiled the significance of InsR activation in mediating BRD7's effect on glucose homeostasis in the liver. Moreover, we identified an interaction between BRD7 and InsR. Notably, our findings indicate that IRS1/2 is not necessary for BRD7's regulation of glucose metabolism, particularly in the context of obesity. The upregulation of hepatic BRD7 significantly reduces blood glucose levels and restores glucose homeostasis in high-fat diet-challenged liver-specific IRS1/2 knockout mice. These findings highlight the presence of an alternative insulin signaling pathway that operates independently of IRS1/2 and offer novel insights into the mechanisms of a previously unknown insulin signaling in obesity.

Genome-wide association study identifies multiple HLA loci for sarcoidosis susceptibility.

Sarcoidosis is a complex systemic disease. Our study aimed to (1) identify novel alleles associated with sarcoidosis susceptibility; (2) provide an in-depth evaluation of HLA alleles and sarcoidosis susceptibility and (3) integrate genetic and transcription data to identify risk loci that may more directly impact disease pathogenesis. We report a genome-wide association study of 1335 sarcoidosis cases and 1264 controls of European descent (EA) and investigate associated alleles in a study of African Americans (AA: 1487 cases and 1504 controls). The EA and AA cohort was recruited from multiple United States sites. HLA alleles were imputed and tested for association with sarcoidosis susceptibility. Expression quantitative locus and colocalization analysis were performed using a subset of subjects with transcriptome data. Forty-nine SNPs in the HLA region in HLA-DRA, -DRB9, -DRB5, -DQA1 and BRD2 genes were significantly associated with sarcoidosis susceptibility in EA, rs3129888 was also a risk variant for sarcoidosis in AA. Classical HLA alleles DRB1*0101, DQA1*0101 and DQB1*0501, which are highly correlated, were also associated with sarcoidosis. rs3135287 near HLA-DRA was associated with HLA-DRA expression in peripheral blood mononuclear cells and bronchoalveolar lavage from subjects and lung tissue and whole blood from GTEx. We identified six novel SNPs (out of the seven SNPs representing the 49 significant SNPs) and nine HLA alleles associated with sarcoidosis susceptibility in the largest EA population. We also replicated our findings in an AA population. Our study reiterates the potential role of antigen recognition and/or presentation HLA class II genes in sarcoidosis pathogenesis.

MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease affecting motor neurons and characterized by microglia-mediated neurotoxic inflammation whose underlying mechanisms remain incompletely understood. In this work, we reveal that MAPK/MAK/MRK overlapping kinase (MOK), with an unknown physiological substrate, displays an immune function by controlling inflammatory and type-I interferon (IFN) responses in microglia which are detrimental to primary motor neurons. Moreover, we uncover the epigenetic reader bromodomain-containing protein 4 (Brd4) as an effector protein regulated by MOK, by promoting Ser492-phospho-Brd4 levels. We further demonstrate that MOK regulates Brd4 functions by supporting its binding to cytokine gene promoters, therefore enabling innate immune responses. Remarkably, we show that MOK levels are increased in the ALS spinal cord, particularly in microglial cells, and that administration of a chemical MOK inhibitor to ALS model mice can modulate Ser492-phospho-Brd4 levels, suppress microglial activation, and modify the disease course, indicating a pathophysiological role of MOK kinase in ALS and neuroinflammation.

Molecular Dynamics Simulations Combined with Markov Model to Explore the Effect of Allosteric Inhibitor Binding on Bromodomain-Containing Protein 4

Bromodomain-Containing Protein 4 (BRD4) can play an important role in gene transcriptional regulation of tumor development and survival by participating in histone modification epigenetic mechanism. Although it has been reported that novel allosteric inhibitors such as ZL0590 have a high affinity with target protein BRD4 and good efficacy, their inhibitory mechanism has not been studied further. The aim of this study was to reveal the inhibition mechanism of allosteric inhibitor ZL0590 on Free-BRD4 and BRD4 binding MS436 (orthosteric inhibitor) by molecular dynamics simulation combined with a Markov model. Our results showed that BRD4-ZL0590 led to α-helices formation of 100–105 compared with Free-BRD4; the combination of MS436 caused residues 30–40 and 95–105 to form α-helices, while the combination of allosteric inhibitors untangled the α-helices formed by the MS436. The results of Markov flux analysis showed that the binding process of inhibitors mainly involved changes in the degree of α-helices at ZA loop. The binding of ZL0590 reduced the distance between ZA loop and BC loop, blocked the conformation at the active site, and inhibited the binding of MS436. After the allosteric inhibitor binding, the MS436 that could normally penetrate into the interior of the pocket was floating on the edge of the active pocket and did not continue to penetrate into the active pocket as expected. In summary, we provide a theoretical basis for the inhibition mechanism of ZL0590 against BRD4, which can be used as a reference for improving the development of drug targets for cancer therapy.

Clinicopathological and gene mutation characteristics of uterine carcinosarcoma

To explore the clinicopathological characteristics, immunophenotype, diagnosis and differential diagnosis of uterine carcinosarcoma (UCS), and to explore the gene mutation characteristics and tumor mutation burden (TMB) of UCS. The clinical imaging, pathomorphological data and immunohistochemical expression of 4 cases of UCS, which were archived in the Department of Pathology of the Second Affiliated Hospital of Soochow University from January 2021 to May 2022 were retrospectively analyzed. All exon groups of 4 cases of UCS were sequenced. All the 4 patients were female, aged 47-81 years. The maximum diameter of the tumor was 4.0-13.0 cm, and the boundary was unclear. Microscopically, the tumor was composed of malignant epithelium and sarcoma. Immunohistochemistry showed that the epithelial components of 4 patients expressed broad-spectrum cytokeratin (AE1/E3), the sarcoma components expressed Vimentin, PAX8, ER, PR were expressed to varying degrees, and Ki-67 positive index was high (60%-90%). There were 3 p53 missense mutations, 1 nonsense mutation, 4 MLH1, PMS2, MSH2, MSH6 were positive and PD-L1 was negative. The sequencing results of the whole exon group of 4 UCS patients showed that TP53, BCL9L, BRD4, CLTCLI, PSMD1I, PLEC genes showed a high mutation ratio, which was 3/4, 2/4, 2/4, 2/4, 2/4, 2/4, respectively. TMB analysis showed that the TMB of 4 cases of UCS was<5 mut/Mb. UCS is a rare and highly malignant endometrial tumor. The sequencing results of the whole exon group suggested that TP53, BCL9L, BRD4 and other genes had high mutation rates, suggesting that the occurrence and development of UCS may be closely related to Wnt signaling pathway. Molecular typing indicated that 3 cases of UCS were of high copy number type/p53 mutation type, and 1 case had POLD1 mutation. Microsatellite stability, low PD-L1 expression and TMB results suggested that UCS patients have no obvious advantage in immunotherapy.

Bromodomain-containing Protein 4 regulates innate inflammation via modulation of alternative splicing.

Bromodomain-containing Protein 4 (BRD4) is a transcriptional regulator which coordinates gene expression programs controlling cancer biology, inflammation, and fibrosis. In the context of airway viral infection, BRD4-specific inhibitors (BRD4i) block the release of pro-inflammatory cytokines and prevent downstream epithelial plasticity. Although the chromatin modifying functions of BRD4 in inducible gene expression have been extensively investigated, its roles in post-transcriptional regulation are not well understood. Given BRD4's interaction with the transcriptional elongation complex and spliceosome, we hypothesize that BRD4 is a functional regulator of mRNA processing. To address this question, we combine data-independent analysis - parallel accumulation-serial fragmentation (diaPASEF) with RNA-sequencing to achieve deep and integrated coverage of the proteomic and transcriptomic landscapes of human small airway epithelial cells exposed to viral challenge and treated with BRD4i. We discover that BRD4 regulates alternative splicing of key genes, including Interferon-related Developmental Regulator 1 (IFRD1) and X-Box Binding Protein 1 (XBP1), related to the innate immune response and the unfolded protein response (UPR). We identify requirement of BRD4 for expression of serine-arginine splicing factors, splicosome components and the Inositol-Requiring Enzyme 1 IREα affecting immediate early innate response and the UPR. These findings extend the transcriptional elongation-facilitating actions of BRD4 in control of post-transcriptional RNA processing via modulating splicing factor expression in virus-induced innate signaling.

Identification of BRD7 by whole-exome sequencing as a predictor for intermediate-stage hepatocellular carcinoma in patients undergoing TACE.

In the present study, we aimed to identify potential predictors of intermediate-stage hepatocellular carcinoma (HCC) using whole-exome sequencing (WES) in patients undergoing transarterial chemoembolization (TACE). In A total of 51 patients, newly diagnosed with intermediate-stage HCC between January 2013 and December 2020, were enrolled. Prior to treatment, histological samples were collected for western blotting and immunohistochemistry. The predictive roles of clinical indicators and genes in patient prognosis were analyzed using univariate and multivariate analyses. Finally, the correlation between imaging features and gene signatures was examined. Using WES, we identified that bromodomain-containing protein 7 (BRD7) was significantly mutated in patients with different TACE responses. No significant difference in BRD7 expression was observed between patients with and without BRD7 mutations. HCC tumors exhibited higher BRD7 than normal liver tissues. Multivariate analysis revealed that alpha-fetoprotein (AFP), BRD7 expression, and BRD7 mutations were independent risk factors for progression-free survival (PFS). In addition, Child-Pugh class, BRD7 expression, and BRD7 mutations were independent risk factors for overall survival (OS). Patients with wild-type BRD7 and high BRD7 expression had worse PFS and OS, whereas those with mutated BRD7 and low BRD7 expression exhibited the best PFS and OS. The Kruskal-Wallis test revealed that wash-in enhancement on computed tomography might be an independent risk factor for high BRD7 expression. BRD7 expression may be an independent risk factor for prognosis in patients with HCC undergoing TACE. Imaging features such as wash-in enhancement are closely related to BRD7 expression.

Nanovesicles loaded with a TGF-β receptor 1 inhibitor overcome immune resistance to potentiate cancer immunotherapy

The immune-excluded tumors (IETs) show limited response to current immunotherapy due to intrinsic and adaptive immune resistance. In this study, it is identified that inhibition of transforming growth factor-β (TGF-β) receptor 1 can relieve tumor fibrosis, thus facilitating the recruitment of tumor-infiltrating T lymphocytes. Subsequently, a nanovesicle is constructed for tumor-specific co-delivery of a TGF-β inhibitor (LY2157299, LY) and the photosensitizer pyropheophorbide a (PPa). The LY-loaded nanovesicles suppress tumor fibrosis to promote intratumoral infiltration of T lymphocytes. Furthermore, PPa chelated with gadolinium ion is capable of fluorescence, photoacoustic and magnetic resonance triple-modal imaging-guided photodynamic therapy, to induce immunogenic death of tumor cells and elicit antitumor immunity in preclinical cancer models in female mice. These nanovesicles are further armored with a lipophilic prodrug of the bromodomain-containing protein 4 inhibitor (i.e., JQ1) to abolish programmed death ligand 1 expression of tumor cells and overcome adaptive immune resistance. This study may pave the way for nanomedicine-based immunotherapy of the IETs.

Exploring the chemical space of functionalized [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the bromodomain of BRD9

Here we report a detailed structure–activity relationship (SAR) study related to [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the reader module of bromodomain containing-protein 9 (BRD9). 3D structure-based pharmacophore models, previously introduced by us, were here employed to evaluate a second generation of compounds, exploring different substitution patterns on the heterocyclic core. Starting from the promising data obtained from our previously identified [1,2,4]triazolo[4,3-a]quinoxaline-based compounds 1–4, the combination of in silico studies, chemical synthesis, biophysical and in vitro assays led to the identification of a new set of derivatives, selected for thoroughly exploring the chemical space of the bromodomain binding site. In more details, the investigation of different linkers at C-4 position highlighted the amine spacer as mandatory for the binding with the protein counterpart and the crucial role of the alkyl substituents at C-1 for increasing the selectivity toward BRD9. Additionally, the importance of a hydrogen bond donor group, critical to anchor the ZA region and required for the interaction with Ile53 residue, was inferred from the analysis of our collected results. Herein we also propose an optimization and an update of our previously reported “pharm-druglike2” 3D structure-based pharmacophore model, introducing it as “pharm-druglike2.1”. Compounds 24-26, 32, 34 and 36 were identified as new valuable BRD9 binders featuring IC50 values in the low micromolar range. Among them, 24 and 36 displayed an excellent selectivity towards BRD9 and a good antiproliferative effect on a panel of leukemia models, especially toward CCRF-CEM cell line, with no cytotoxicity on healthy cells. Notably, the interaction of 24 and 36 with the bromodomain and PHD finger-containing protein 1 (BRPF1) also emerged, disclosing them as new and unexplored dual inhibitors for these two proteins highly involved in leukemia. These findings highlight the potential for the identification of new attractive dual epidrugs as well as a promising starting point for the development of chemical degraders endowed with anticancer activities.

Near-Infrared-Activatable PROTAC Nanocages for Controllable Target Protein Degradation and On-Demand Antitumor Therapy.

As a novel protein knockdown tool, proteolysis targeting chimeras (PROTACs) can induce potent degradation of target proteins by hijacking E3 ubiquitin ligases. However, the uncontrollable protein disruption of PROTACs is prone to cause "off-target" toxicity after systemic administration. Herein, we designed a photocaged-PROTAC (phoBET1) and loaded it in UCNPs-based mesoporous silica nanoparticles (UMSNs) to construct a NIR light-activatable PROTAC nanocage (UMSNs@phoBET1) for controllable target protein degradation. Upon NIR light (980 nm) irradiation, UMSNs@phoBET1 nanocages could be activated to release active PROTAC via a controlled pattern for degrading bromodomain-containing protein 4 (BRD4) and inducing MV-4-11 cancer cell apoptosis. In vivo experiments demonstrated that UMSNs@phoBET1 nanocages were capable of responding to NIR light in tumor tissues to achieve BRD4 degradation and effectively suppress tumor growth. This NIR light-activatable PROTAC nanoplatform compensates for the current shortcomings of short-wavelength light-controlled PROTACs and presents a paradigm for the precise regulation of PROTACs in living tissues.

Structural insights of novel mutational frames in Bromodomain Containing-2 gene (BRD2) in juvenile myoclonic epilepsy: bed, bench, and laptop profiles

PurposeJuvenile myoclonic epilepsy (JME) is an adolescent onset type of idiopathic generalized epilepsies. Bromodomain containing protein-2 gene (BRD2), a transcriptional regulatory protein, has a susceptible role in the expression of JME. Considering the polymorphic variations observed in exon 3 of the BRD2 gene, we evaluated the molecular interactions with anti-seizure medication in individuals diagnosed with JME. MethodsThe genomic DNA was extracted from 5 mL of peripheral venous blood of JME participants (n = 55) and healthy control subjects (n = 55). Detailed anti-seizure medication and outcomes were noted during the study period. Identified novel mutations at nucleotide and protein sequences, compared by multiple sequence alignment. Wild-type (WT) and mutated-type (MT) structures were investigated for molecular docking and interactions with anti-seizure drugs. ResultsA common variant at c.1707G>A was found among 23 participants, while a single variant at c.1663ins C was found in one participant. The deletion positions were observed at c.1890delA, c.1892A>T, c.1895A>T, c.1896G>T, c.1897T>C, c.1898T>C, c.1899C>T, c.1900G>T, c.1901C>T and c.1902A>T exhibiting stop codon after p.111Pro>stop; these variants resulted in a truncated protein. In silico analysis was conducted to validate changes; docking analysis showed that novel variant has a significant role in the interactions with anti-seizure drugs. SignificanceBesides clinical and genetic outcomes, ∼5.45% unique genetical variations were observed in the participants. Significant mimicked at the binding site position (92–111) of human BRD2 ranges ∼8.2%, ∼16.4%, and ∼10.6%. Further, research is needed to identify the importance of polymorphism alterations at the binding site and their molecular interactions with anti-seizure drugs, which might be confirmed in a diverse population with JME.

Downregulation of BRD4 attenuates high glucose-induced damage of trophoblast cells by inhibiting activation of AKT/mTOR pathway.

It was elucidated that bromodomain-containing protein 4 (BRD4) has involvement with diabetic complication. However, the role and molecular mechanism of BRD4 in gestational diabetes mellitus (GDM) are still unclear. In this study, the mRNA and protein contents of BRD4 in placenta tissues of GDM patients and high glucose (HG)-induced HTR8/SVneo cells were detected by qRT-PCR and western blot assay. CCK-8, EdU staining, flow cytometry as well as western blot were applied for the appraisement of cell viability and apoptosis. Wound healing assay and transwell assay were conducted for the assessment of cell migration and invasion. Oxidative stress and inflammatory factors were detected. Additionally, the contents of AKT/mTOR pathway-related proteins were estimated applying western blot. It was discovered that BRD4 expression was ascended in tissues and HG-induced HTR8/SVneo cells. BRD4 downregulation cut down the contents of p-AKT and p-mTOR but had no effects on the total protein levels of AKT or mTOR in HG-induced HTR8/SVneo cells. BRD4 depletion promoted cell viability, enhanced proliferative capability, and reduced cell apoptotic level. Moreover, BRD4 depletion facilitated cell migrative and invasive capabilities, and repressed the oxidative stress as well as inflammatory damage in HG-induced HTR8/SVneo cells. The activation of Akt reversed the protective impacts of BRD4 depletion on HG-induced HTR8/SVneo cells. To sum up, BRD4 silencing may alleviate HG-induced HTR8/SVneo cell damage through the inhibition of the AKT/mTOR pathway.

Novel RNA epigenetics-driven technologies for rapid prediction of cancer drug resistance.

e19001 Background: The human genome comprises ~99% non-coding DNA sequences (ncDNAs), most of which are actively transcribed into various forms of non-coding RNAs (ncRNAs) that undergo extensive chemical modifications to regulate various functions of the subcellular organelles in human cells ( Statello, et al. Nat Rev Mol Cell Biol 2020). However, the clinical potential of RNA modifications and RNA modifying proteins, i.e., RNA epigenetics, is largely unexplored. In a recent study ( Cheng et al. Nat Commun 2018), we first demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, directly interact with elongating RNA polymerase II (eRNAPII) and the bromodomain-containing protein 4 (BRD4) to form distinct active chromatin structures (ACS) that regulate drug resistance in leukemia cells. This proof-of-concept study enabled us to develop the first-generation of RNA epigenetics-driven drug-predictive system. The primary goal of this study is to validate and optimize this novel drug-predictive system with clinical myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Methods: We developed new technologies, including 5-ethynyl uridine click chemistry (EC) and proximity ligation rolling cycle amplification (PL-RCA)-coupled confocal microscopy (CM), flow cytometry (FCM) and RNA sequencing (RNA-seq). Immunohistochemistry (IHC), western blot (WB), co-immunoprecipitation (co-IP), and dot blot were also used in this study. Results: Our data demonstrated significantly increased expression of the NSUN1, NSUN2 and other key components of the drug-resistant ACS in MDS/AML, which is correlated with disease progression and drug resistance in MDS and AML. We have developed novel technologies, i.e., EC- and PL-RCA-coupled CM and FCM (EC-/PL-RCA/CM &amp; FCM) to visualize and quantify RCMTs-mediated ACS and nascent RNA synthesis in MDS/AML. We discovered nascent RNA synthesis as a highly sensitive and super-fast drug-predictive marker that is independent of cell death and apoptosis. Our data demonstrated that PL-RCA/FCM and EC-/CM have a very high sensitivity to detect the NSUN1/2-mediated ACS in leukemia cell lines and clinical MDS/AML specimens. Our preliminary data demonstrated that increased levels of NSUN1/2-mediated ACS detected by PL-RCA/FCM in pre-treatment MDS/AML can rapidly predict the responses to venetoclax and/or azacitidine. We are actively pursuing RNA-seq targeting NSUN1/2-associated nascent RNAs and ACS, which provides a novel strategy to dissect the functional genomics and RNA epigenetics underlying cancer drug resistance. Conclusions: We have developed a novel RCMTs/NSUN1/2-targeting system for rapid prediction and in-depth analysis of drug resistance in MDS/AML. A large sized clinical study is needed to further validate its usefulness in clinical settings.

Design of Class I/IV Bromodomain-Targeting Degraders for Chromatin Remodeling Complexes.

Targeted protein degradation is an emerging technology that can be used for modulating the activity of epigenetic protein targets. Among bromodomain-containing proteins, a number of degraders for the BET family have been developed, while non-BET bromodomains remain underexplored. Several of these proteins are subunits in chromatin remodeling complexes often associated with oncogenic roles. Here, we describe the design of class I (BPTF and CECR2) and IV (BRD9) bromodomain-targeting degraders based on two scaffolds derived from pyridazinone and pyrimidine-based heterocycles. We evaluate various exit vectors and linkers to identify analogues that demonstrate selectivity within these families. We further use an in-cell NanoBRET assay to demonstrate that these heterobifunctional molecules are cell-permeable, form ternary complexes, and can degrade nanoluciferase-bromodomain fusions. As a first example of a CECR2 degrader, we observe that our pyrimidine-based analogues degrade endogenous CECR2 while showing a smaller effect on BPTF levels. The pyridazinone-based compounds did not degrade BPTF when observed through Western blotting, further supporting a more challenging target for degradation and a goal for future optimization.

Current progress in the targeted therapy of breast cancer: Structure-activity correlation and docking studies (2015-2021).

Despite cancer research and therapy, breast cancer remains a complicated health crisis in women and represents a top biomedical research priority. Nowadays, breast cancer is an extremely heterogeneous disease and is known as the leading cause of death among women worldwide. The incidence and mortality rates of breast cancer have been increasing gradually for the past decades. Nowadays, common treatments for breast cancer are chemotherapy, endocrine therapy, immunotherapy, radiotherapy, and surgery. The most common targets in breast cancer treatment are human epidermal growth factor receptor 2 (HER2) and estrogen receptors. The literature suggests that several targets/pathways are also involved in the development of breast cancer, that is, poly(ADP-ribose) polymerase (PARP), bromodomain-containing protein 4 (BRD4), cyclin-dependent kinase 4/6 (CDK4/6), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), polo-like kinase 1 (PLK1), phosphoinositide 3-kinases/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR), histone deacetylase (HDAC), nuclear factor kappa B (NF-κB), PD-L1, and aromatase inhibitors. Meanwhile, the study of breast cancer is a hot topic in the current scenario of basic/clinical research. This review article provides information on different targets associated with breast cancer and summarizes the progress of current research on synthesized inhibitors as anti-breast cancer agents from 2015 to 2021. The review aims to provide structure-activity relationship and docking studies for designing novel compounds for breast cancer therapy.

LncRNA GAS5 regulated by FTO-mediated m6A demethylation promotes autophagic cell death in NSCLC by targeting UPF1/BRD4 axis.

Long non-coding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) has been shown to be a regulator for many cancers, including non-small cell lung cancer (NSCLC). Therefore, its role and mechanism in the process of NSCLC deserve to be further revealed. The expression levels of GAS5, fat mass and obesity-associated protein (FTO) and bromodomain-containing protein 4 (BRD4) were detected by quantitative real-time PCR. Western blot analysis was used to examine the protein expression of FTO, BRD4, up-frameshift protein 1 (UPF1) and autophagy-related markers. Methylated RNA immunoprecipitation was used to assess the m6A level of GAS5 regulated by FTO. Cell proliferation and apoptosis were determined using MTT assay, EdU assay and flow cytometry. Autophagy ability was assessed by immunofluorescence staining and transmission electron microscope. Xenograft tumor model was constructed to explore the effects of FTO and GAS5 on NSCLC tumor growth in vivo. The interaction between UPF1 and GAS5 or BRD4 was confirmed by pull-down assay, RIP assay, dual-luciferase reporter assay, and chromatin immunoprecipitation. Fluorescent in situ hybridization was used to analyze the co-localization of GAS5 and UPF1. Actinomycin D treatment was employed to evaluate BRD4 mRNA stability. GAS5 was downregulated in NSCLC tissues and was associated with poor prognosis in NSCLC patients. FTO was highly expressed in NSCLC, and it inhibited GAS5 expression by reducing GAS5 m6A methylation level. GAS5 suppressed by FTO could promote the autophagic death of NSCLC cells in vitro and inhibit NSCLC tumor growth in vivo. In addition, GAS5 was able to interact with UPF1 to reduce the mRNA stability of BRD4. Knockdown of BRD4 reversed the inhibition of GAS5 or UPF1 silencing on the autophagic cell death of NSCLC. The findings of the study showed that lncRNA GAS5 mediated by FTO could contribute to the autophagic cell death of NSCLC by interacting with UPF1 to reduce BRD4 mRNA stability, suggesting that GAS5 might be a vital therapy target for NSCLC progression.

Development of versatile solid-phase methods for syntheses of PROTACs with diverse E3 ligands

Developing highly active proteolysis-targeting chimeras (PROTACs) requires investigating a variety of ubiquitin ligase (E3 ligase) ligands and linker structures as well as their lengths. In this study, we developed a solid-phase synthesis method that affords PROTAC design diversity. We expanded the E3 ligand range to include Von Hippel-Lindau (VHL) and inhibitor of apoptosis protein (IAP) ligands because only the cereblon (CRBN) ligand thalidomide and its derivatives have been investigated for solid-phase synthesis of PROTACs. Moreover, we examined the suitability of a polyethylene glycol (PEG) rather than an alkyl linker used in our previous study for synthesizing PROTACs. Facile and rapid solid-phase synthesis methods using the above E3 ligands for developing PROTACs targeting bromodomain-containing protein 4 (BRD4) were accomplished. Western blotting analysis revealed that minor differences in the E3 ligand and linker type significantly affected the activity of the synthesized PROTACs. Our solid-phase PROTAC synthesis methods enable rapid synthesis of multiple PROTACs with various combinations of ligands for the protein-of-interest and E3 ligands and linkers that connect these ligands.

The Role of Bromodomain-Containing Proteins in Development and Disease

The Role of Bromodomain-Containing Proteins in Development and Disease

Orchestrating epigenetic readers: Progress in understanding the functions of bromodomain-containing protein 4 complexes

Orchestrating epigenetic readers: Progress in understanding the functions of bromodomain-containing protein 4 complexes