Ann F. Hoffman

Leukotriene B4 receptor antagonists as therapeutics for inflammatory disease: preclinical and clinical developments

Leukotriene B4 (LTB4) is a lipid inflammatory mediator derived from membrane phospholipids by the sequential actions of cytosolic phospholipase A2 (PLA2), 5-lipoxygenase (5-LO) and leukotriene A4 (LTA4) hydrolase. Several inflammatory diseases, including asthma, chronic obstructive pulmonary disease, arthritis and inflammatory bowel disease, have been associated with elevated levels of LTB4. As a result, pharmacological strategies to modulate the synthesis of LTB4 (inhibition of PLA2, 5-LO or LTA4 hydrolase) or the effects of LTB4 itself (antagonism of LTB4 receptors) are being developed by several companies. Two G-protein-coupled receptors mediate the effects of LTB4, namely BLT1 and BLT2. The pharmacology, expression and function of these two receptors were last reviewed by Tager and Luster in 2004. Since then, there has been an increased understanding of the function of these receptors, in particular for the lesser understood of the two receptors, BLT2. Furthermore, since last reviewed in 1996, there have been several clinical developments in the use of BLT receptor antagonists for inflammatory diseases. This review summarizes the latest preclinical and clinical developments in BLT antagonism for inflammatory diseases and discusses potential future developments.

BET Bromodomain Proteins Mediate Downstream Signaling Events following Growth Factor Stimulation in Human Lung Fibroblasts and Are Involved in Bleomycin-Induced Pulmonary Fibrosis

Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain–containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor–responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.

High-throughput confocal microscopy for β-arrestin-green fluorescent protein translocation G protein-coupled receptor assays using the evotec opera

Ligand-activated G protein-coupled receptors (GPCRs) are known to regulate a myriad of homeostatic functions. Inappropriate signaling is associated with several pathophysiological states. GPCRs belong to a approximately 800 member superfamily of seven transmembrane-spanning receptor proteins that respond to a diversity of ligands. As such, they present themselves as potential points of therapeutic intervention. Furthermore, orphan GPCRs, which are GPCRs without a known cognate ligand, offer new opportunities as drug development targets. This chapter describes a systems-based biological approach, one that combines in silico bioinformatics, genomics, high-throughput screening, and high-content cell-based confocal microscopy strategies to (1) identify a relevant subset of protein family targets, (2) within the therapeutic area of energy metabolism/obesity, (3) and to identify small molecule leads as tractable combinatorial and medicinal chemistry starting points. Our choice of screening platform was the Transfluor beta-arrestin-green fluorescent protein translocation assay in which full-length human orphan GPCRs were stably expressed in a U-2 OS cell background. These cells lend themselves to high-speed confocal imaging techniques using the Evotec Technologies Opera automated microscope system. The basic assay system can be implemented in any laboratory using a fluorescent probe, a stably expressed GPCR of interest, automation-assisted plate and liquid-handling techniques, an optimized image analysis algorithm, and a high-speed confocal microscope with sophisticated data analysis tools.

A Pharmaceutical Company User’s Perspective on the Potential of High Content Screening in Drug Discovery

It is early to fully reflect on the state of the art in high content screening (HCS), because it is still a relatively new approach in drug discovery. Although the development of the first microscopes are a century old and the first confocal microscope is only 20 yr old, the fluorescent probes used within HCS along with the combination of robotic automation and integrated software technologies are quite new. HCS will require a few more years to fully demonstrate its potential power in drug discovery. Within the last year, however, one has seen this ever-expanding field lure participants in from all areas of science, introducing newer versions of instruments and reagents such that the combined efforts result in platforms and tools that meet many organizational goals in multiple ways. The potential of HCS today lies in its versatility. HCS can be used for primary screening, basic research, target identification, biomarkers, cytotoxicity, and helping to predict clinical outcomes. HCS is being applied to stem cells, patient cells, primary hepatocytes, and immortalized cultured cells. We have noted for individual specialized assays, there are multiple solutions just as there are for those standardized universally accepted assays. Whether we have needed to query cellular processes under live conditions or wanted to follow kinetically the course of a compounds effects on particular cellular reactions, we have been hampered by only a few limitations. This chapter offers a glimpse inside the use of HCS in our drug discovery environment.

Inactivation of Mirk/Dyrk1B Kinase Targets Quiescent Pancreatic Cancer Cells

A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G0 tumor cells. Mirk uses several mechanisms to block cell cycling, and Mirk increases expression of antioxidant genes that decrease reactive oxygen species (ROS) levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G0 state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1, and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S-phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In previous studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase. Mol Cancer Ther; 10(11); 2104–14. ©2011 AACR.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

Abstract 649: Mirk/dyrk1B kinase inactivation increases gemcitabine efficacy towards quiescent pancreatic cancer cells

A major problem in the treatment of cancer is the presence of residual quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such cancer cells are believed to cause tumor recurrence when they re-enter the cell cycle under favorable conditions of the microenvironment. Gemcitabine is the drug most often used for treatment of pancreatic cancer, but the response rate is less than 20%. Since stratagems to increase delivery of gemcitabine by inhibiting stromal hedgehog signaling have shown some success, identifying methods to increase gemcitabine efficacy is timely. The serine/threonine kinase Mirk/dyrk1B is expressed at very low levels in most normal tissues, but is overexpressed or amplified in most pancreatic cancers. Mirk maintains pancreatic cancer cells in a viable quiescent state through its transcriptional co-activator functions for a set of antioxidant proteins that decrease intracellular levels of reactive oxygen species (Cancer Res 2009;69:3317-24). We now show that Panc1 and SU86.86 pancreatic cancer cells undergo a reversible quiescent arrest in G0 through activation of Chk2 and autophagy to reduce ribosomal RNA levels, with little apoptosis or senescence. Depletion of Mirk/dyrk1B by either induced shRNA or transfected RNAi duplexes or inhibition of Mirk9s kinase activity by a small molecule inhibitor increased DNA damage in quiescent pancreatic cancer cells, either grown attached to petri dishes or in small clumps in suspension as a model for ascites. DNA damage was detected by increased phosphorylation of the histone protein H2AX and by an increase in S phase checkpoints after the quiescent cells re-entered cycle in response to serum mitogens. Pharmacological inhibition of Mirk kinase induced a dose-dependent cleavage of the apoptotic marker proteins PARP and caspase 3, reduced Akt activation, and led to cleavage of the autophagic marker protein LC3. Pretreatment with the Mirk kinase inhibitor increased tumor cell kill 5-fold by short exposures to marginally toxic gemcitabine concentrations. In prior studies Mirk depletion had no detectable effect on the viability of normal diploid fibroblasts (Cancer Res 2007;67:7247-55), while other investigators have shown that embryonic knockout was not lethal (Leder et al, Biochem J 2003;372:881-8). Thus the small molecule Mirk/dyrk1B kinase inhibitor sensitized pancreatic cancer cells to low gemcitabine concentrations, while knockout or depletion of Mirk/dyrk1B had no detectable effect on normal tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 649. doi:10.1158/1538-7445.AM2011-649