Targeting MAP3K19 prevents human lung myofibroblast activation both in vitro and in a humanized SCID model of idiopathic pulmonary fibrosis.

Idiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-ß pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.

MAP3K19 Is Overexpressed in COPD and Is a Central Mediator of Cigarette Smoke-Induced Pulmonary Inflammation and Lower Airway Destruction.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung inflammation resulting in a progressive decline in lung function whose principle cause is cigarette smoke. MAP3K19 is a novel kinase expressed predominantly by alveolar and interstitial macrophages and bronchial epithelial cells in the lung. We found that MAP3K19 mRNA was overexpressed in a limited sampling of lung tissue from COPD patients, and a closer examination found it to be overexpressed in bronchoalveolar macrophages from COPD patients, as well as the bronchial epithelium and inflammatory cells in the lamina propria. We further found MAP3K19 to be induced in various cell lines upon environmental stress, such as cigarette smoke, oxidative and osmotic stress. Exogenous expression of MAP3K19 in cells caused an upregulation of transcriptionally active NF-κB, and secretion of the chemokines CXCL-8, CCL-20 and CCL-7. Inhibition of MAP3K19 activity by siRNA or small molecular weight inhibitors caused a decrease in cigarette smoke-induced inflammation in various murine models, which included a decrease in pulmonary neutrophilia and KC levels. In a chronic cigarette smoke model, inhibition of MAP3K19 significantly attenuated emphysematous changes in airway parenchyma. Finally, in a viral exacerbation model, mice exposed to cigarette smoke and influenza A virus showed a decrease in pulmonary neutrophilia, pro-inflammatory cytokines and viral load upon inhibition of MAP3K19. Collectively, these results suggest that inhibition of MAP3K19 may represent a novel strategy to target COPD that promises to have a potential therapeutic benefit for patients.

MAP3K19 Is a Novel Regulator of TGF-ß Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-ß has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-ß-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-ß stimulation. TGF-ß-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.

A new perspective of cannabinoid 1 receptor antagonists: approaches toward peripheral CB1R blockers without crossing the blood-brain barrier.

Since Rimonabant was withdrawn in Europe in 2008 because of its substantial CNS risk factors including depression and anxiety, the development of anti-obesity drugs targeting CB1R in the brain has been suspended and/or terminated globally. Instead, developing peripherally restricted CB1R antagonists is actively pursued in the hope that not only could they eliminate any CNS adverse effects observed with Rimonabant, but also maintain therapeutic benefits in metabolic syndrome, including obesity, type 2 diabetes, and non-alcoholic fatty liver diseases. In this review, we summarized the most recent advances that have been made on this area, with particular emphasis on various synthetic approaches, whereby the increase in polarity, water solubility and polar surface area were centralized on, toward potential peripheral-acting CB1 antagonists.

A concise total synthesis of (+/-)-acutifolone A.

Starting from 4-methylcyclohexanone (7), a concise total synthesis of the pinguisane-type sesquiterpenoid acutifolone A, in racemic form, has been accomplished in 14 steps with an overall yield of 14.5%.

Murine bone marrow-derived mast cells express chemoattractant receptor-homologous molecule expressed on T-helper class 2 cells (CRTh2).

Mast cells are bone marrow-derived effector cells that can initiate inflammatory responses to infectious organisms or allergens by releasing a multitude of pro-inflammatory factors including prostaglandin (PG) D(2). We demonstrate that primary murine bone marrow-derived mast cells (BMMCs) express the PGD(2) receptor; chemoattractant receptor-homologous molecule expressed on T(h) class 2 cells (CRT(h)2). Activation of CRT(h)2 on BMMC by PGD(2) or the CRT(h)2-specific agonist, 13,14-dihydro-15-keto-prostaglandin D(2) (DK-PGD(2)), resulted in signaling response including Ca(2+) mobilization and phosphorylation of the p42/p44 extracellular signal-regulated kinases (ERKs) kinases. Phosphorylation of the ERKs could be blocked by pertussis toxin, as well as a small molecule antagonist of CRT(h)2, Compound A. Activation of CRT(h)2 on BMMC also resulted in the up-regulation of CD23 and CD30 on the cell surface, as well as CD62L shedding. Finally, PGD(2) and DK-PGD(2) induced the migration of BMMC in vitro and in vivo in response to an intra-dermal DK-PGD(2) injection. Both these processes were inhibited by the CRT(h)2 antagonist. These results raise the possibility that the functional consequences of the PGD(2)-CRT(h)2 interaction on mast cells may be relevant in allergic inflammation.

Polyene cyclization promoted by the cross-conjugated alpha-carbalkoxy cyclohexenone system. An unusual 1,2-hydride shift under Lewis acid catalysis.

Polyene cyclization of the titled compounds under catalysis with AlCl(3)/SnCl(4) gave rise to the corresponding polycyclic products, many of which were structurally highly unexpected, and thus, individual X-ray analysis was required to finalize the structural identification. Mechanistically, an unusual 1,2-hydride shift is proposed to elucidate the product formation.

A small molecule CRTH2 antagonist inhibits FITC-induced allergic cutaneous inflammation.

A FITC-induced allergic contact hypersensitivity model was used to investigate the role that the prostaglandin D(2) receptor-chemoattractant receptor-homologous molecule expressed on T(h)2 cells (CRTH2) plays in modulating cutaneous inflammation. Our results show that inhibition of CRTH2, achieved via administration of a potent, small molecule antagonist, Compound A (Cmpd A), effectively blocked edema formation and greatly reduced the inflammatory infiltrate and skin pathology observed in drug vehicle-treated animals. Gene expression analysis revealed that Cmpd A administration down-regulated the transcription of a wide range of pro-inflammatory mediators. This correlated with decreases in cytokine and chemokine protein levels, notably IL-4, IL-1beta, tumor necrosis factor-alpha, transforming growth factor-beta, GRO-alpha, MIP-2 and thymic stromal lymphopoietin (TSLP) in FITC-challenged ears. The administration of an anti-TSLP-neutralizing antibody was only partially effective in lowering the FITC-induced inflammatory infiltrate and cytokine production compared with the CRTH2 antagonist. Taken together, these data suggest that blockade of CRTH2 inhibits multiple pathways leading to cutaneous inflammation in this model. This suggests that CRTH2 antagonism may be a viable route for therapeutic intervention in allergic skin diseases, such as atopic dermatitis.

Antagonism of CRTH2 ameliorates chronic epicutaneous sensitization-induced inflammation by multiple mechanisms.

Prostaglandin D(2) (PGD(2)) and its receptor chemoattractant receptor homologous molecule expressed on T(h)2 cells (CRTH2) have been implicated in the pathogenesis of numerous allergic diseases. We investigated the role of PGD(2) and CRTH2 in allergic cutaneous inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated cutaneous inflammation caused by either repeated epicutaneous ovalbumin or FITC sensitization. Gene expression and ELISA analysis revealed that there was reduced pro-inflammatory cytokine mRNA or protein produced. Importantly, the CRTH2 antagonist reduced total IgE, as well as antigen-specific IgE, IgG1 and IgG2a antibody levels. This reduction in antibody production correlated to reduced cytokines produced by splenocytes following in vitro antigen challenge. An examination of skin CD11c(+) dendritic cells (DC) showed that in mice treated with the CRTH2 antagonist, there was a decrease in the number of these cells that migrated to the draining lymph nodes in response to FITC application to the skin. Additionally, naive CD4(+) T lymphocytes co-cultured with skin-derived DC from CRTH2 antagonist-treated mice showed a reduced ability to produce a number of cytokines compared with DC from vehicle-treated mice. Collectively, these findings suggest that CRTH2 has a pivotal role in mediating the inflammation and the underlying immune response following epicutaneous sensitization.

CRTH2 antagonism significantly ameliorates airway hyperreactivity and downregulates inflammation-induced genes in a mouse model of airway inflammation.

Prostaglandin D(2), the ligand for the G protein-coupled receptors DP1 and CRTH2, has been implicated in the pathogenesis of the allergic response in diseases such as asthma, rhinitis, and atopic dermatitis. This prostanoid also fulfills a number of physiological, anti-inflammatory roles through its receptor DP1. We investigated the role of PGD(2) and CRTH2 in allergic pulmonary inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated inflammation caused by either acute or subchronic sensitization using the cockroach egg antigen. Gene expression and ELISA analysis revealed that there was reduced proinflammatory cytokine mRNA or protein produced, as well as a wide array of genes associated with the Th2-type proinflammatory response. Importantly, the CRTH2 antagonist reduced antigen-specific IgE, IgG1, and IgG2a antibody levels as well as decreased mucus deposition and leukocyte infiltration in the large airways. Collectively, these findings suggest that the PGD(2)-CRTH2 activation axis has a pivotal role in mediating the inflammation and the underlying immune response in a T cell-driven model of allergic airway inflammation.

Polyene cyclization promoted by the cross-conjugated alpha-carbalkoxy enone system. Observation on a putative 1,5-hydride/1,3-alkyl shift under Lewis acid catalysis.

Polyene cyclization of compounds 3 and 4 under catalysis with AlCl3 and/or SnCl4 gave rise to complex bicyclic products 8 and 9, structures of which were highly unexpected, and X-ray analyses were invoked for unambiguously structural identification. Mechanistically, a tandem sigma-bond rearrangement process, including an unusual through-space 1,5-hydride or 1,3-alkyl shift as a key operation, is proposed.

Small-molecule CRTH2 antagonists for the treatment of allergic inflammation: an overview.

The discovery of a second receptor for prostaglandin D2, chemoattractant receptor-homologous molecule expressed on T helper type 2 (CRTH2), has revived significant efforts into the development of small-molecule antagonists for allergic diseases as the receptor is predominantly expressed on cells such as eosinophils, TH2 cells and basophils, which are major pro-inflammatory cells in diseases such as asthma and allergic rhinitis. This brief review serves to illustrate current patent literature focusing on the similarities and differences of an ever-growing number of CRTH2 antagonists emerging from corporate laboratories.

Application of the Barton photochemical reaction in the synthesis of 1-dethia-3-aza-1-carba-2-oxacephem: a novel agent against resistant pathogenic microorganisms.

Racemic 7-(phenylacetamido)-1-dethia-3-aza-1-carba-2-oxacephem 3 was synthesized and found to possess antibacterial activity against Staphylococcus aureus FDA 209P, Escherichia coli ATCC 39188, Pseudomonas aeruginosa 1101-75 and Klebsiella pneumoniae NCTC 418 as well as the beta-lactamase producing organisms E. coli A9675 and P. aeruginosa 18S-H and the methicillin-resistant organism S. aureus 95. Formation of the carbacephem 3 originated from the Barton photochemical reaction in the conversion of 8 to 10. Intramolecular cyclization of syn-oximino beta-lactam 10 afforded 7-azido-2-oxa-3-azacephem 11, which was reduced and acylated to 12. Enzymatic removal of the methyl group from 12 gave the target molecule 3.

Design, synthesis, and anticancer activity of phosphonic acid diphosphate derivative of adenine-containing butenolide and its water-soluble derivatives of paclitaxel with high antitumor activity.

Synthesis of adenine derivative of triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 4 was accomplished by treatment of phosphonate 3 with 5-phosphoribosyl 1-pyrophosphate in the presence of 5-phosphoribosyl 1-pyrophosphate synthetase. It was found that triphosphonate 4 functions as an irreversible stoichiometric inactivator of the Escherichia coli ribonucleoside diphosphate reductase (RDPR). Triphosphonate 4 exhibited potent inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines. Paclitaxel ester derivatives of adenine-containing triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 8-10 were also synthesized. Like triphosphonate 4, compound 8 exhibited inhibitory property toward RDPR. It also induced microtubule assembly similar to paclitaxel (5). The structure of the chlorodiester linker in 8 was found to account for this dual property. After treatment of MCF7 cells with compounds 4, 5, and 8, fluorescence microscope examination demonstrated the presence of nucleus shrinkage or segmentation. Bifunctional prodrug 8 exhibited higher lipophilicity than 4 and higher water-solubility than 5. Pro-dual-drug 8 exhibited more pronounced anticancer activity relative to that of the triphosphonate 4 and paclitaxel (5). In contrast, compound 9, resulting from the linkage of triphosphonate 4 and paclitaxel (5) through a diester unit, was only found to function as a highly water-soluble prodrug for paclitaxel (5). It induced microtubule assembly in vitro, but did not show inhibitory property toward RDPR. On the other hand, compound 10, an aggregate of triphosphonate 4 and paclitaxel (5), neither functioned as an inhibitor of RDPR nor exhibited microtubule assembly stimulating activity in vitro.

Diels-Alder chemistry of 2-diethoxyphosphinylcyclohex-2-enones. A new approach to complex phosphonates and synthetic applications of the beta-keto phosphonate system.

Enone phosphonates 1 and 2 were found to be excellent dienophiles for the Diels-Alder reaction, giving phosphonate-containing polycycles, and the phosphonate group of the resulting adducts facilitated both the installation of an angular alkyl group via a reductive alkylation process and the regioselective generation of a ring junction double bond via an intramolecular Wadsworth-Horner-Emmons reaction.