Synthesis of Novel Triazine-Based Chalcones and 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines as Potential Leads in the Search of Anticancer, Antibacterial and Antifungal Agents.

This study presents the synthesis of four series of novel hybrid chalcones (20,21)a-g and (23,24)a-g and six series of 1,3,5-triazine-based pyrimido[4,5-b][1,4]diazepines (28-33)a-g and the evaluation of their anticancer, antibacterial, antifungal, and cytotoxic properties. Chalcones 20b,d, 21a,b,d, 23a,d-g, 24a-g and the pyrimido[4,5-b][1,4]diazepines 29e,g, 30g, 31a,b,e-g, 33a,b,e-g exhibited outstanding anticancer activity against a panel of 60 cancer cell lines with GI50 values between 0.01 and 100 µM and LC50 values in the range of 4.09 µM to >100 µM, several of such derivatives showing higher activity than the standard drug 5-fluorouracil (5-FU). On the other hand, among the synthesized compounds, the best antibacterial properties against N. gonorrhoeae, S. aureus (ATCC 43300), and M. tuberculosis were exhibited by the pyrimido[4,5-b][1,4]diazepines (MICs: 0.25-62.5 µg/mL). The antifungal activity studies showed that triazinylamino-chalcone 29e and triazinyloxy-chalcone 31g were the most active compounds against T. rubrum and T. mentagrophytes and A. fumigatus, respectively (MICs = 62.5 µg/mL). Hemolytic activity studies and in silico toxicity analysis demonstrated that most of the compounds are safe.

Binding Mechanism of Inhibitors to BRD4 and BRD9 Decoded by Multiple Independent Molecular Dynamics Simulations and Deep Learning.

Bromodomain 4 and 9 (BRD4 and BRD9) have been regarded as important targets of drug designs in regard to the treatment of multiple diseases. In our current study, molecular dynamics (MD) simulations, deep learning (DL) and binding free energy calculations are integrated to probe the binding modes of three inhibitors (H1B, JQ1 and TVU) to BRD4 and BRD9. The MD trajectory-based DL successfully identify significant functional function domains, such as BC-loop and ZA-loop. The information from the post-processing analysis of MD simulations indicates that inhibitor binding highly influences the structural flexibility and dynamic behavior of BRD4 and BRD9. The results of the MM-GBSA calculations not only suggest that the binding ability of H1B, JQ1 and TVU to BRD9 are stronger than to BRD4, but they also verify that van der Walls interactions are the primary forces responsible for inhibitor binding. The hot spots of BRD4 and BRD9 revealed by residue-based free energy estimation provide target sites of drug design in regard to BRD4 and BRD9. This work is anticipated to provide useful theoretical aids for the development of selective inhibitors over BRD family members.

Synergy of EGFR and AURKA Inhibitors in KRAS-mutated Non-small Cell Lung Cancers.

The most common oncogenic driver mutations for non-small cell lung cancer (NSCLC) activate EGFR or KRAS. Clinical trials exploring treatments for EGFR- or KRAS-mutated (EGFRmut or KRASmut) cancers have focused on small-molecule inhibitors targeting the driver mutations. Typically, these inhibitors perform more effectively based on combination with either chemotherapies, or other targeted therapies. For EGFRmut NSCLC, a combination of inhibitors of EGFR and Aurora-A kinase (AURKA), an oncogene commonly overexpressed in solid tumors, has shown promising activity in clinical trials. Interestingly, a number of recent studies have indicated that EGFR activity supports overall viability of tumors lacking EGFR mutations, and AURKA expression is abundant in KRASmut cell lines. In this study, we have evaluated dual inhibition of EGFR and AURKA in KRASmut NSCLC models. These data demonstrate synergy between the EGFR inhibitor erlotinib and the AURKA inhibitor alisertib in reducing cell viability and clonogenic capacity in vitro, associated with reduced activity of EGFR pathway effectors, accumulation of enhanced aneuploid cell populations, and elevated cell death. Importantly, the erlotinib-alisertib combination also synergistically reduces xenograft growth in vivo. Analysis of signaling pathways demonstrated that the combination of erlotinib and alisertib was more effective than single-agent treatments at reducing activity of EGFR and pathway effectors following either brief or extended administration of the drugs. In sum, this study indicates value of inhibiting EGFR in KRASmut NSCLC, and suggests the specific value of dual inhibition of AURKA and EGFR in these tumors. SIGNIFICANCE: The introduction of specific KRAS G12C inhibitors to the clinical practice in lung cancer has opened up opportunities that did not exist before. However, G12C alterations are only a subtype of all KRAS mutations observed. Given the high expression of AURKA in KRASmut NSCLC, our study could point to a potential therapeutic option for this subgroup of patients.

BET inhibitor nanotherapy halts kidney damage and reduces chronic kidney disease progression after ischemia-reperfusion injury.

Targeting epigenetic mechanisms has emerged as a potential therapeutic approach for the treatment of kidney diseases. Specifically, inhibiting the bromodomain and extra-terminal (BET) domain proteins using the small molecule inhibitor JQ1 has shown promise in preclinical models of acute kidney injury (AKI) and chronic kidney disease (CKD). However, its clinical translation faces challenges due to issues with poor pharmacokinetics and side effects. Here, we developed engineered liposomes loaded with JQ1 with the aim of enhancing kidney drug delivery and reducing the required minimum effective dose by leveraging cargo protection. These liposomes efficiently encapsulated JQ1 in both the membrane and core, demonstrating superior therapeutic efficacy compared to freely delivered JQ1 in a mouse model of kidney ischemia-reperfusion injury. JQ1-loaded liposomes (JQ1-NPs) effectively targeted the kidneys and only one administration, one-hour after injury, was enough to decrease the immune cell (neutrophils and monocytes) infiltration to the kidney-an early and pivotal step to prevent damage progression. By inhibiting BRD4, JQ1-NPs suppress the transcription of pro-inflammatory genes, such as cytokines (il-6) and chemokines (ccl2, ccl5). This success not only improved early the kidney function, as evidenced by decreased serum levels of BUN and creatinine in JQ1-NPs-treated mice, along with reduced tissue expression of the damage marker, NGAL, but also halted the production of extracellular matrix proteins (Fsp-1, Fn-1, α-SMA and Col1a1) and the fibrosis development. In summary, this work presents a promising nanotherapeutic strategy for AKI treatment and its progression and provides new insights into renal drug delivery.

Disruption of Super-Enhancers in Activated Pancreatic Stellate Cells Facilitates Chemotherapy and Immunotherapy in Pancreatic Cancer.

One major obstacle in the drug treatment of pancreatic ductal adenocarcinoma (PDAC) is its highly fibrotic tumor microenvironment, which is replete with activated pancreatic stellate cells (a-PSCs). These a-PSCs generate abundant extracellular matrix and secrete various cytokines to form biophysical and biochemical barriers, impeding drug access to tumor tissues. Therefore, it is imperative to develop a strategy for reversing PSC activation and thereby removing the barriers to facilitate PDAC drug treatment. Herein, by integrating chromatin immunoprecipitation (ChIP)-seq, Assays for Transposase-Accessible Chromatin (ATAC)-seq, and RNA-seq techniques, this work reveals that super-enhancers (SEs) promote the expression of various genes involved in PSC activation. Disruption of SE-associated transcription with JQ1 reverses the activated phenotype of a-PSCs and decreases stromal fibrosis in both orthotopic and patient-derived xenograft (PDX) models. More importantly, disruption of SEs by JQ1 treatments promotes vascularization, facilitates drug delivery, and alters the immune landscape in PDAC, thereby improving the efficacies of both chemotherapy (with gemcitabine) and immunotherapy (with IL-12). In summary, this study not only elucidates the contribution of SEs of a-PSCs in shaping the PDAC tumor microenvironment but also highlights that targeting SEs in a-PSCs may become a gate-opening strategy that benefits PDAC drug therapy by removing stromal barriers.

Discovery of novel biaryl benzoxazepinones as dual-mode receptor-interacting protein kinase-1 (RIPK1) inhibitors.

Systemic inflammatory response syndrome (SIRS), an exaggerated defense response of the organism to a noxious stressor, involves a massive inflammatory cascade that ultimately leads to reversible or irreversible end-organ dysfunction and even death. Suppressing RIPK1, a key protein in necroptosis pathway, has been proven to be an effective therapeutic strategy for inflammation and SIRS. In this study, a series of novel biaryl benzoxazepinone RIPK1 inhibitors were designed and synthesized by introducing different aryl substituents at the C7 position of benzoxazepinone. As a result, p-cyanophenyl substituted analog 19 exhibited the most potent in vitro anti-necroptotic effect in HT-29 cells (EC50 = 1.7 nM) and superior protection against temperature loss and death in mice in the TZ-induced SIRS model compared to GSK'772. What's more, in vivo analysis of the levels of inflammatory factors in mice also revealed that compound 19 had better anti-inflammatory activity than GSK'772.

A bromodomain-independent mechanism of gene regulation by the BET inhibitor JQ1: direct activation of nuclear receptor PXR.

Bromodomain and extraterminal (BET) proteins are extensively studied in multiple pathologies, including cancer. BET proteins modulate transcription of various genes, including those synonymous with cancer, such as MYC. Thus, BET inhibitors are a major area of drug development efforts. (+)-JQ1 (JQ1) is the prototype inhibitor and is a common tool to probe BET functions. While showing therapeutic promise, JQ1 is not clinically usable, partly due to metabolic instability. Here, we show that JQ1 and the BET-inactive (-)-JQ1 are agonists of pregnane X receptor (PXR), a nuclear receptor that transcriptionally regulates genes encoding drug-metabolizing enzymes such as CYP3A4, which was previously shown to oxidize JQ1. A PXR-JQ1 co-crystal structure identified JQ1's tert-butyl moiety as a PXR anchor and explains binding by (-)-JQ1. Analogs differing at the tert-butyl lost PXR binding, validating our structural findings. Evaluation in liver cell models revealed both PXR-dependent and PXR-independent modulation of CYP3A4 expression by BET inhibitors. We have characterized a non-BET JQ1 target, a mechanism of physiological JQ1 instability, a biological function of (-)-JQ1, and BET-dependent transcriptional regulation of drug metabolism genes.

Failure of the dual orexin receptor antagonist suvorexant to engender drug discrimination in rats.

For abuse potential assessment, U.S. Food and Drug Administration (FDA) requests that new, brain-penetrating drugs are ideally compared with approved drugs that share the mechanism of action and are judged to have abuse liability by the Drug Enforcement Agency. For development of the dual orexin receptor antagonist (DORA) daridorexant, the FDA recommended conducting a rat drug discrimination paradigm against the approved, schedule IV, DORA suvorexant. Surprisingly, at suvorexant plasma levels up to three-fold the maximum concentration at the highest approved human dose, rats did not learn to discriminate the suvorexant stimulus from vehicle.

A Novel Aldisine Derivative Exhibits Potential Antitumor Effects by Targeting JAK/STAT3 Signaling.

The JAK/STAT3 signaling pathway is aberrantly hyperactivated in many cancers, promoting cell proliferation, survival, invasiveness, and metastasis. Thus, inhibitors targeting JAK/STAT3 have enormous potential for cancer treatment. Herein, we modified aldisine derivatives by introducing the isothiouronium group, which can improve the antitumor activity of the compounds. We performed a high-throughput screen of 3157 compounds and identified compounds 11a, 11b, and 11c, which contain a pyrrole [2,3-c] azepine structure linked to an isothiouronium group through different lengths of carbon alkyl chains and significantly inhibited JAK/STAT3 activities. Further results showed that compound 11c exhibited the optimal antiproliferative activity and was a pan-JAKs inhibitor capable of inhibiting constitutive and IL-6-induced STAT3 activation. In addition, compound 11c influenced STAT3 downstream gene expression (Bcl-xl, C-Myc, and Cyclin D1) and induced the apoptosis of A549 and DU145 cells in a dose-dependent manner. The antitumor effects of 11c were further demonstrated in an in vivo subcutaneous tumor xenograft experiment with DU145 cells. Taken together, we designed and synthesized a novel small molecule JAKs inhibitor targeting the JAK/STAT3 signaling pathway, which has predicted therapeutic potential for JAK/STAT3 overactivated cancer treatment.

Design of an Ante-enhancer with an Azone-Mimic Structure using Ionic Liquid.

PURPOSE: Laurocapram (Azone) was broadly examined as a representative enhancer of skin penetration in the 1980s. However, it was not approved for treatment because it caused skin irritation following its penetration into the epidermis through the stratum corneum. In the present study, a so-called ante-enhancer with an Azone-mimic structure was designed based on an ante-drug with negligible systemic toxic effects following its permeation through the skin. METHODS: The ante-enhancer was designed using ionic liquid technology: an ionic liquid-type ante-enhancer (IL-Azone) with an Azone-mimic structure was prepared from ε-caprolactam and myristic acid as cationic and anionic substances, respectively. The enhancing effects of IL-Azone on the permeation by the following model drugs through pig skin were examined: isosorbide 5-mononitrate (ISMN), antipyrine (ANP), and fluorescein isothiocyanate dextran (FD-4). Skin irritation by IL-Azone was assessed using the Draize method. RESULTS: The primary irritation index (P.I.I.) of IL-Azone by the Draize method was markedly lower than that of Azone (6.9). Although the ability of IL-Azone to enhance skin penetration was not as high as Azone, IL-Azone moderately increased skin permeation by the model compounds tested (ISMN: 4.7 fold, ANP: 4.5 fold, FD-4: 4.0 fold). CONCLUSIONS: These results suggest the usefulness of designing a skin penetration enhancer using ionic liquid technology. Further trials on the ionic liquid design with an Azone-mimic structure using other cations and anions may lead to the development of better ante-enhancers.

Effectiveness of suvorexant versus benzodiazepine receptor agonist sleep drugs in reducing the risk of hip fracture: Findings from a regional population-based cohort study.

Sleep drugs are often necessary to treat insomnia in older patients. Benzodiazepine receptor agonists (BZRAs) are primarily used for insomnia in these patients, but there are concerns regarding their association with delirium and bone fractures. Among sleep drugs, orexin receptor antagonists such as suvorexant have a lower risk of delirium than BZRAs, but their effectiveness in preventing hip fractures is unknown. Hip fracture is a life-threatening trauma in advanced-age patients and a social problem. Therefore, we investigated the relationship between suvorexant and hip fracture. The Shizuoka Kokuho Database was used to compare the time to hip fracture in patients who had been newly taking suvorexant and other sleep drugs such as benzodiazepines since November 2014. A proportional hazards model for hip fracture as an outcome was used to estimate the hazard ratio. Propensity scores were estimated using a logistic regression model, and the confounding factors were age, sex, several comorbidities, and each oral medication. The suvorexant group comprised 6860 patients (110 with hip fracture), and the BZRA group (benzodiazepines and Z-drugs) comprised 50,203 patients (1487 with hip fracture). In the matched cohort (6855:6855 patients), 259 and 249 patients in the suvorexant and BZRA group developed hip fractures during the observational period, respectively. The hazard ratio of the suvorexant group compared with the BZRA group was 1.48 (95% confidence interval, 1.20-1.82). In the subgroup analysis, patients in the suvorexant group had a higher risk of hip fracture if they were aged >75 years, had no diabetes, had no neurological disease, had no renal failure, had liver disease, had hypertension, were not taking alpha 1 blockers, and were not taking oral steroids. Among people in the Japanese regional population who use sleep drugs, patients taking suvorexant can be at higher risk of hip fracture than patients taking BZRAs.

Synthesis of pyrido-annelated [1,2,4,5]tetrazines, [1,2,4]triazepine, and [1,2,4,5]tetrazepines for anticancer, DFT, and molecular docking studies.

In this strategy, we attempt to design various novel nitrogen-rich heterocycles in one molecule. Green, simple, and efficient aza-annulations of an active, versatile building block, 1-amino-4-methyl-2-oxo-6-phenyl-1,2-dihydropyridine-3-carbonitrile (1), with different bifunctional reagents were developed under solvent-free conditions, resulting in the bridgehead tetrazines and azepines (triazepine and tetrazepines). Pyrido[1,2,4,5]tetrazines have been synthesized through two pathways; [3 + 3]- and [5 + 1]-annulations. In addition, pyrido-azepines have been developed by applying [4 + 3]-and [5 + 2]-annulations. This protocol establishes an efficient technique for synthesizing essential biological derivatives of 1,2,4,5-tetrazines, 1,2,4-triazepines, and 1,2,4,5-tetrazepine, tolerating a diverse variety of functionalities without the need for catalysis and fast reaction rates in high yields. The National Cancer Institute (NCI, Bethesda, USA) examined twelve compounds produced at a single high dosage (10-5 M). Compounds 4, 8, and 9 were discovered to have potent anticancer action against certain cancer cell types. To explain NCI results, the density of states was calculated to conduct a better description of the FMOs. The molecular electrostatic potential maps were created to explain a molecule's chemical reactivity. In silico ADME experiments were performed to better understand their pharmacokinetic characteristics. Finally, the molecular docking investigations on Janus Kinase-2 (PDB ID: 4P7E) were carried out to study the binding mechanism, binding affinity, and non-bonding contacts.

Synthesis and biological evaluation of 1-phenyl-4,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(1H)-one/thiones as anticancer agents.

Cancer is associated with uncontrolled cell proliferation invading adjoining tissues and organs. Despite the availability of several chemotherapeutic agents, the constant search for newer approaches and drugs is necessitated owing to the ever-growing challenge of resistance. Over the years, DNA has emerged as an important druggable therapeutic drug due to its role in critical cellular processes such as cell division and maintenance. Further, evading apoptosis stands out as a hallmark of cancer. Hence, designing new compounds that would target DNA and induce apoptosis plays an important role in cancer therapy. In the current work, we carried out the synthesis and anticancer evaluation of 1-aryl-4,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(1H)-ones/thiones (26 compounds) against selected human cancer cell lines. Among these, compounds 8ae, 8ad, 8cf, 10ad and Kenpaullone have shown good inhibitory properties against HeLa cells (IC50 < 2 µM) with good selectivity over the non-cancerous human embryonic kidney (Hek293T) cells. In cell cycle analysis, the compounds 8ad and 8cf have exhibited G2/M cell cycle arrest in HeLa cells. In addition, the compounds 8ad and 8cf induced apoptosis in a dose-dependent manner in the Annexin-V FITC staining assay. The DAPI staining clearly demonstrated the condensed and fragmented nuclei in 8ad, 8cf, 8ae and Kenpaullone-treated HeLa cells. In addition, these compounds strongly suppressed the healing after 48 h in in vitro cell migration assay. The DNA binding experiments indicated that compounds 8ae, 8cf, and 8ad as well as Kenpaullone interact with double-stranded DNA by binding in grooves which may interrupt the DNA replication and kill fast-growing cells. Molecular docking studies revealed the binding pose of 8ad and Kenpaullone at HT1 binding pocket of double-stranded DNA. Compounds 8ad and 8cf demonstrated moderate topo II inhibition which could be a possible reason for their anticancer properties. Compounds 8ad and 8cf may cause the topo II and DNA covalent complex, which leads to the inhibition of DNA replication and transcription. This eventually increases the DNA damage in cells and promotes cell apoptosis. With the above interesting biological profile, the new 1-aryl-2,6-dihydrobenzo[b]pyrazolo[3,4-d]azepin-5(4H)-one/thione derivatives have emerged as promising leads for the discovery of new anticancer agents.

Regulation of inflammatory genes in decidual cells: Involvement of the bromodomain and extra-terminal family proteins.

The decidua undergoes proinflammatory activation in late pregnancy, promoting labor. Bromodomain and Extra-Terminal (BET) family proteins interact with acetylated histones and may control gene expression in inflammation. Here, we assessed whether BETs are involved in inflammatory gene regulation in human decidual cells. We have treated primary cultures of decidual stromal cells (DSCs) from term pregnancies with endotoxin (LPS) and measured the expression of a panel of pro-and anti-inflammatory genes. BET involvement was assessed using the selective BET inhibitors (+)-JQ1 and I-BET-762 or the negative control compound (-)-JQ1. Histone 3 and -4 acetylation and BETs binding at the target gene promoters were determined to assess whether these processes are involved in the actions of LPS, BETs, and BET inhibitors. LPS increased the expression of the proinflammatory (PTGS2, IL6, CXCL8/IL8, TNF) and the anti-inflammatory (IL10, IDO1) genes of the panel. The constitutively expressed inflammatory genes (PTGS1, PTGES) were unaffected. The BET inhibitors, but not the control compound, reduced the basal and LPS-induced expression of PTGS1, PTGS2, IL6, CXCL8/IL8, IL10, and IDO1. TNF expression was not changed by BET inhibition. The dominant BETs were Bromodomain-containing protein -2 (BRD2) and -4L (BRD4L) in DSCs. LPS increased histone 4 acetylation at the CXCL8/IL8 and TNF promoters and histone 3 and -4 acetylation at the IDO1 promoter, while (+)-JQ1 abrogated histone acetylation at several promoters. Overall, histone acetylation and promoter binding of BETs showed no consistent relationship with gene expression across the gene panel and the treatments. BET proteins, predominantly BRD2 and BRD4L, control critical pro- and anti-inflammatory genes in DSCs. TNF induction exemplifies a BET-independent pathway. Changing histone acetylation at the promoters is not a general obligatory requirement for inflammatory gene expression in response to LPS. BETs likely act at chromatin loci separate from the examined promoters. BET inhibitors may block decidual activation at labor.

Synthesis, biological activity and mechanism of action of novel allosecurinine derivatives as potential antitumor agents.

Cancer with low survival rates is the second main cause of death among all diseases in the world and consequently, effective antineoplastic agents are urgently needed. Allosecurinine is a plant-derived indolicidine securinega alkaloid shown bioactivity. The object of this study is to investigate synthetic allosecurinine derivatives with considerable anticancer capacity against nine human cancer cell lines as well as mechanism of action. We synthesized twenty-three novel allosecurinine derivatives and evaluated their antitumor activity against nine cancer cell lines for 72 h by MTT and CCK8 assays. FCM was applied to analyze the apoptosis, mitochondrial membrane potential, DNA content, ROS production, CD11b expression. Western blot was selected to analyze the protein expression. Structure-activity relationships were established and potential anticancer lead BA-3 which induced differentiation of leukemia cells towards granulocytosis at low concentration and apoptosis at high concentration was identified. Mechanism studies showed that mitochondrial pathway mediated apoptosis within cancer cells with cell cycle blocking was induced by BA-3. In addition, western blot assays revealed that BA-3 induced expression of the proapoptotic factor Bax, p21 and reduced the levels of antiapoptotic protein such as Bcl-2, XIAP, YAP1, PARP, STAT3, p-STAT3, and c-Myc. Collectively, BA-3 was a lead compound for oncotherapy at least in part, through the STAT3 pathway. These results were an important step in further studies on allosecurinine-based antitumor agent development.

Co-administration of JQ1, a bromodomain-containing protein 4 inhibitor, enhances the antitumor effect of combretastatin A4, a microtubule inhibitor, while attenuating its cardiotoxicity.

Combretastatin A4 (CA4) inhibits microtubule polymerization, and clinical trials of the prodrug, CA4 disodium phosphate (CA4DP), as an anti-cancer agent have been conducted. However, CA4DP has not been marketed to date because the margin between the effective dose and the cardiotoxic dose is insufficient. Meanwhile, bromodomain-containing protein 4 (BRD4) has been reported to be required for recovery from mitotic arrests induced by anti-microtubule drugs. BRD4 has also been reported to be involved in the progression of heart failure. Therefore, we hypothesized that the combined use of CA4DP with BRD4 inhibitors can enhance the antitumor effect and attenuate CA4DP-induced cardiotoxicity. In this study, the antitumor effect and cardiotoxicity caused by the co-administration of CA4DP with JQ1, a BRD4 inhibitor, were evaluated. CA4 or JQ1 alone reduced the viability of cultured canine mammary tumor cells (CHMp-13a). Viability was further reduced by co-administration, through the suppression of c-Myc. BRD4 positivity in CHMp-13a cytoplasm showed a significant increase when treated with CA4 alone, while the increase was not significant following co-administration. In CHMp-13a xenograft-transplanted mice, co-administration of CA4DP and JQ1 suppressed tumor growth significantly. In CA4DP-induced cardiac injury model rats, echocardiography showed a CA4DP-induced decrease in cardiac function and histopathology showed cardiomyocyte necrosis. Meanwhile, these cardiac changes tended to be milder following the co-administration of CA4DP and JQ1. These results suggest that CA4DP-JQ1 co-administration enhances the antitumor effect of CA4DP while attenuating its cardiotoxicity and therefore potentially open the doors to the development of a novel cancer chemotherapy with reduced cardiotoxicity risks.

The epigenetic regulator BRD4 is required for myofibroblast differentiation of knee fibroblasts.

Arthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra-terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA-seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT-qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFß1-induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA-mediated depletion of BRD4. Together, these data reveal that BRD4-mediated epigenetic events support TGFß1-mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.

Design and synthesis of novel rigid dibenzo[b,f]azepines through ring closure technique as promising anticancer candidates against leukaemia and acting as selective topoisomerase II inhibitors and DNA intercalators.

In this research, two novel series of dibenzo[b,f]azepines (14 candidates) were designed and synthesised based on the rigidification principle and following the reported doxorubicin's pharmacophoric features. The anti-proliferative activity was evaluated at the NCI against a panel of 60 cancer cell lines. Further, the promising candidates (5a-g) were evaluated for their ability to inhibit topoisomerase II, where 5e was noticed to be the most active congener. Moreover, its cytotoxicity was evaluated against leukaemia SR cells. Also, 5e arrested the cell cycle at the G1 phase and increased the apoptosis ratio by 37.34%. Furthermore, in vivo studies of 5e showed the inhibition of tumour proliferation and the decrease in its volume. Histopathology and liver enzymes were examined as well. Besides, molecular docking, physicochemical, and pharmacokinetic properties were carried out. Finally, a SAR study was discussed to open the gate for further optimisation of the most promising candidate (5e).HighlightsTwo novel series of dibenzo[b,f]azepines were designed and synthesised based on the rigidification principle in drug design.The anti-proliferative activity was evaluated at the NCI against a panel of 60 cancer cell lines.5e was the most active anti-topo II congener (IC50 = 6.36 ± 0.36 µM).5e was evaluated against leukaemia SR cells and its cytotoxic effect was confirmed (IC50 = 13.05 ± 0.62 µM).In vivo studies of 5e significantly inhibited tumour proliferation by 62.7% and decreased tumour volume to 30.1 mm3 compared to doxorubicin treatment.

Bromodomain Protein BRD4-Mediated Mutant p53 Transcription Promotes TNBC Progression.

TP53 is the most common mutated gene in human cancer. Mutant p53 protein loses its tumor-suppressor properties and gains oncogenic activity. Mutant p53 is a therapeutic target in a broad range of cancer types. However, how mutant p53 is epigenetically regulated during tumor progression remains elusive. In this study, we found that the upregulation of mutant p53 is mediated by bromodomain protein BRD4 in triple-negative breast cancer (TNBC) cells. Inhibition of BRD4 with its inhibitor JQ1 or knockdown of BRD4 suppressed the transcription of mutant p53, which led to the re-expression of p21, the inhibition of S-phase entry, and colony formation in TNBC cells. BRD4 also positively regulated the transcription of wild-type p53, whereas JQ1 treatment and knockdown of BRD4 decreased the expression of p21 in MCF-7 cells. Knockdown of BRD4 resulted in attenuation of TNBC tumor growth in vivo. Taken together, our results uncover a novel regulatory mechanism of mutant p53 via BRD4, and suggest that the bromodomain inhibitor suppresses tumorigenesis through targeting mutant p53 in TNBC.