gsk525762a and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

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.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Arthritis; Cell Cycle Proteins; Disease Models, Animal; Half-Life; Humans; Kinetics; Mice; Neoplasms; Quinazolines; Structure-Activity Relationship; Transcription Factors

The symptoms of HIV-sensory neuropathy are dominated by neuropathic pain. Recent data show that repeated use of opiates enhances the chronic pain states in HIV patients. Limited attention has so far been devoted to exploring the exact pathogenesis of HIV painful disorder and opiate abuse in vivo, for which there is no effective treatment. Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal domain protein (BET) family and functions as a chromatin 'reader' that binds acetylated lysines in histones in brain neurons to mediate the transcriptional regulation underlying learning and memory. Here, we established a neuropathic pain model of interaction of intrathecal HIV envelope glycoprotein 120 (gp120) and chronic morphine in rats. The combination of gp120 and morphine (gp120/M, for 5 days) induced persistent mechanical allodynia compared with either gp120 or morphine alone. Mechanical allodynia reached the lowest values at day 10 from gp120/M application, beginning to recover from day 21. In the model, gp120/M induced overexpression of Brd4 mRNA and protein at day 10 using RT-qPCR and western blots, respectively. Immunohistochemical studies showed that Brd4 at day 10 was expressed in the neurons of spinal cord dorsal horn. BET inhibitor I-BET762 dose-dependently increased the mechanical threshold in the gp120/M pain state. The present study provides preclinical evidence for treating HIV neuropathic pain with opioids using the BET inhibitor.

Topics: Analysis of Variance; Animals; Benzodiazepines; CD11b Antigen; Disease Models, Animal; Glial Fibrillary Acidic Protein; HIV Envelope Protein gp120; Hyperalgesia; Male; Morphine; Neuralgia; Nuclear Proteins; Pain Threshold; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord; Transcription Factors; Up-Regulation

Bromodomain inhibitors (JQ1 and I-BET 762) are a new generation of selective, small molecule inhibitors that target BET (bromodomain and extra terminal) proteins. By impairing their ability to bind to acetylated lysines on histones, bromodomain inhibitors interfere with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell cycle, apoptosis and inflammation. In this study, JQ1 and I-BET 762 decreased c-Myc and p-Erk 1/2 protein levels and inhibited proliferation in pancreatic cancer cells. The tumor microenvironment is known to play an important role in pancreatic cancer, and these drugs suppressed the production of nitric oxide and a variety of inflammatory cytokines, including IL-6, CCL2, and GM-CSF, in both immune and pancreatic cancer cells in vitro. Notably, the bromodomain inhibitors also reduced protein levels of p-Erk 1/2 and p-STAT3 in mouse models of pancreatic cancer. All of these proteins are essential for tumor promotion, progression and metastasis. In conclusion, the bromodomain inhibitors JQ1 and I-BET 762 targeted and suppressed multiple pathways in pancreatic cancer. I-BET 762 and a number of other bromodomain inhibitors are currently being tested in several clinical trials, making them potentially promising drugs for the treatment of pancreatic cancer, an often-fatal disease.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Azepines; Benzodiazepines; Cell Line, Tumor; Cell Proliferation; Ceruletide; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Humans; Inflammation Mediators; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide; Pancreatic Neoplasms; Pancreatitis; Phosphorylation; Proto-Oncogene Proteins c-myc; RAW 264.7 Cells; Signal Transduction; Triazoles

Inhibitors of the bromodomain and extraterminal domain family (BETI) have recently entered phase I clinical trials. In patients with advanced leukemia's, potent antileukemia activity was displayed with minimum dose-limiting toxicity. In preclinical models of hematological malignancies, including aggressive B-cell lymphomas, BETI induced cell-cycle arrest and apoptosis. However, the underlying cell death mechanisms are still not well understood. Dissecting the mechanisms required by BETI to mediate cell death would provide strong direction on how to best utilize BETI to treat patients with aggressive hematological malignancies. Herein, we provide understanding of the molecular mechanisms underlying BETI-mediated cell death using I-BET762. Induction of cell death occurred in primary murine and human B-cell lymphomas through apoptosis. Genetic dissection using Eμ-myc B-cell lymphoma compound mutants demonstrated that I-BET762-induced apoptosis does not require the p53 pathway. Furthermore, deletion of Apaf1, and thus the absence of a functional apoptosome, is associated with a delayed drug response but do not provide long-term resistance. Prolonged treatment of this model in fact fails to suppress the therapeutic efficacy of the drug and is associated with biochemical features of autophagy. However, lack of mitochondrial permeability completely inhibited I-BET762-mediated tumor cell death, indicating mitochondrial damage as key events for its activity. Combination of I-BET762 with BH3-only mimetics ABT-263 or obatoclax, restored sensitivity to I-BET762 lymphoma killing; however, success was determined by expression of Bcl-2 family antiapoptotic proteins. Our study provides critical insight for clinical decisions regarding the appropriate strategy for using BETI as a single agent or in combination to treat patients with aggressive B-cell lymphomas.

Topics: Aniline Compounds; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Benzodiazepines; Cell Line, Tumor; Cytoprotection; Disease Models, Animal; Drug Resistance, Neoplasm; Humans; Lymphoma, B-Cell; Mice; Mitochondria; Rituximab; Signal Transduction; Sulfonamides; Tumor Suppressor Protein p53

Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common 'core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain 'super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.

Topics: Animals; Benzodiazepines; Cell Cycle Proteins; Cell Line, Tumor; Disease Models, Animal; Drug Evaluation, Preclinical; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Mice; Nuclear Proteins; Nucleophosmin; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Xenograft Model Antitumor Assays

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzodiazepines; Cell Cycle Proteins; Cell Proliferation; Cluster Analysis; Disease Models, Animal; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Gene Silencing; Humans; Kinetics; Mice; Models, Molecular; Molecular Conformation; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; RNA-Binding Proteins; Signal Transduction; Transcription Factors; Tumor Burden; Xenograft Model Antitumor Assays

MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.

Topics: Animals; Antineoplastic Agents; Azepines; Benzodiazepines; Cell Line, Tumor; Disease Models, Animal; Drug Discovery; Humans; Mice; Multiple Myeloma; Nuclear Proteins; Protein Structure, Tertiary; Proto-Oncogene Proteins c-myc; Transcriptional Activation; Triazoles