Characterisation and Profiling of Standard Atopic Dermatitis Therapies in a Chronic Dermatitis Murine Model Induced by Oxazolone.

Atopic dermatitis (AD) is a common inflammatory skin disorder characterised by hypersensitivity to allergens, eczematous lesions and pruritus. The aim of this study was to comprehensively characterise a murine model of dermatitis and assess the similarity with the human disease, as well as to profile clinically relevant AD therapies. Four repeated topical administrations of oxazolone in the auricular skin of sensitised mice induced morphological features compatible with AD, including redness and swelling, as well as histological changes typical of spongiotic (eczematous) dermatitis and increased plasmatic IgE.

Identification of novel small molecule-based strategies of COL7A1 upregulation and readthrough activity for the treatment of recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease caused by loss of function mutations in the gene coding for collagen VII (C7) due to deficient or absent C7 expression. This disrupts structural and functional skin architecture, leading to blistering, chronic wounds, inflammation, important systemic symptoms affecting the mouth, gastrointestinal tract, cornea, and kidney function, and an increased skin cancer risk. RDEB patients have an extremely poor quality of life and often die at an early age.

Phenotypic drug discovery: recent successes, lessons learned and new directions.

Many drugs, or their antecedents, were discovered through observation of their effects on normal or disease physiology. For the past generation, this phenotypic drug discovery approach has been largely supplanted by the powerful but reductionist approach of modulating specific molecular targets of interest. Nevertheless, modern phenotypic drug discovery, which combines the original concept with modern tools and strategies, has re-emerged over the past decade to systematically pursue drug discovery based on therapeutic effects in realistic disease models.

Application of a phenotypic drug discovery strategy to identify biological and chemical starting points for inhibition of TSLP production in lung epithelial cells.

Thymic stromal lymphopoietin (TSLP) is a cytokine released by human lung epithelium in response to external insult. Considered as a master switch in T helper 2 lymphocyte (Th2) mediated responses, TSLP is believed to play a key role in allergic diseases including asthma. The aim of this study was to use a phenotypic approach to identify new biological and chemical starting points for inhibition of TSLP production in human bronchial epithelial cells (NHBE), with the objective of reducing Th2-mediated airway inflammation.

Combined anti CXC receptors 1 and 2 therapy is a promising anti-inflammatory treatment for respiratory diseases by reducing neutrophil migration and activation.

Neutrophil infiltration and activation in the lung are important pathophysiological features in COPD, severe asthma and bronchiectasis mostly mediated by CXCL8 and CXCL1 via CXCR1 and CXCR2. No thorough study to date has been performed to compare the anti-inflammatory effect profile of dual CXCR1/2 vs. selective CXCR2 antagonists in relevant human neutrophil assays and pulmonary inflammation models.

Discovery of LAS101057: A Potent, Selective, and Orally Efficacious A2B Adenosine Receptor Antagonist.

The structure-activity relationships for a series of pyrazine-based A2B adenosine receptor antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious A2B receptor antagonist, was identified as a clinical development candidate. LAS101057 inhibits agonist-induced IL-6 production in human fibroblasts and is active in an ovalbumin (OVA)-sensitized mouse model after oral administration, reducing airway hyperresponsiveness to methacholine, Th2 cytokine production, and OVA-specific IgE levels.

Discovery of potent and selective bicyclic A(2B) adenosine receptor antagonists via bioisosteric amide replacement.

Several new potent and selective A(2B) adenosine receptor antagonists have been prepared in which the aryl-amide moiety of the lead series, exemplified by 1a, has been replaced by bioisosteric bicyclic moieties. Although the majority of compounds had generally improved microsomal stability as compared to 1a, this was not translated into overall improvements in the pharmacokinetic profiles of a representative set of compounds.

Discovery of N-(5,6-diarylpyridin-2-yl)amide derivatives as potent and selective A(2B) adenosine receptor antagonists.

The synthesis and SAR of a series of N-(5,6-diarylpyridin-2-yl)amide derivatives as potent A(2B) adenosine receptor antagonists is described. Several compounds showed good selectivity versus other adenosine receptors. The potent and selective analogue 9 was shown to have good oral bioavailability in the rat.

CODES, a novel procedure for ligand-based virtual screening: PDE7 inhibitors as an application example.

Phosphodiesterase (PDE) 7 is a high affinity cAMP-specific PDE whose functional role in T-cells has been the subject of some controversy. Recent findings on tissue distribution, however, support the hypothesis that PDE7 could be a good target for the treatment of airway diseases, T-cell related diseases or central nervous system (CNS) disorders. Therefore, the identification of selective inhibitors targeted against PDE7 enzyme has become an attractive area of research.

Discovery and characterization of 4'-(2-furyl)-N-pyridin-3-yl-4,5'-bipyrimidin-2'-amine (LAS38096), a potent, selective, and efficacious A2B adenosine receptor antagonist.

A novel series of N-heteroaryl 4'-(2-furyl)-4,5'-bipyrimidin-2'-amines has been identified as potent and selective A(2B) adenosine receptor antagonists. In particular, compound 5 showed high affinity for the A(2B) receptor (Ki = 17 nM), good selectivity (IC(50): A(1) > 1000 nM, A(2A) > 2500 nM, A3 > 1000 nM), displayed a favorable pharmacokinetic profile in preclinical species, and showed efficacy in functional in vitro models.

Phosphodiesterase 7A1 is expressed in human CD4+ naïve T cells at higher levels than in CD4+ memory cells and is not required during their CD3/CD28-dependent activation.

PDE7A1 is a cAMP-hydrolyzing phosphodiesterase expressed in lymphoid tissue, where its possible role during T cell activation remains unclear. We have characterized the functional relevance of PDE7A1 in the naïve (CD4+CD45RA+) and memory (CD4+CD45RO+) subsets of human peripheral CD4+ T cells during CD3/CD28-dependent stimulation. Our results indicate that PDE7A1 is expressed in resting naïve CD4+ T cells at higher levels than in the corresponding memory cells and that levels of PDE7A1 mRNA are not upregulated upon CD3/CD28 mediated stimulation of these T cell subsets.

New pyrrolopyrimidin-6-yl benzenesulfonamides: potent A2B adenosine receptor antagonists.

A new series of 4-(1,3-dialkyl-2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)benzenesulfonamides has been identified as potent A2B adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A2B, A1 and A3 adenosine receptors. 6-(4-{[4-(4-Bromobenzyl)piperazin-1-yl]sulfonyl}phenyl)-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione (16) showed a high affinity for the A2B adenosine receptor (IC50=1 nM) and selectivity (A1: 183x; A3: 12660x).

Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits.

Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (alpha or alpha') and two regulatory (beta) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus.

Dynamic localization/association of protein kinase CK2 subunits in living cells: a role in its cellular regulation?

Accumulating evidence indicates that in addition to the classical complex, the catalytic and regulatory subunits of CK2 can also exist as free populations in living cells. The association of recombinant CK2 subunits in vitro has been characterized, providing evidence for the first time for their targeted interactions in living cells. The data also suggest that the regulation by phosphorylation of many CK2 substrates may strongly depend on the dynamic localization/association of its subunits.

Visualization and molecular analysis of nuclear import of protein kinase CK2 subunits in living cells.

We have generated fusion proteins between the subunits of CK2 and GFP and characterized their behaviour in living cells. The expressed fusion proteins were functional and interacted with endogenous CK2. Imaging of NIH3T3 cells expressing low level of GFP-CK2alpha or GFP-CK2beta showed that both proteins were mostly nuclear in interphase.

c-Myc inhibits CD11a and CD11c leukocyte integrin promoters.

The c-Myc transcription factor is an important regulator of cell growth and differentiation, and its gene repression ability seems to play a key role in Myc-mediated cellular transformation. Since Myc overexpression has been associated with reduced expression of beta1 and beta2 integrins, we have investigated the role of c-Myc on CD11a and CD11c transcription. c-Myc inhibited CD11a and CD11c integrin promoter activity in co-transfection experiments, and similar repression was obtained in cells where c-Myc expression (KmycB) or activity (Rat-1 c-MycER) is inducible.

Polyomavirus enhancer-binding protein 2/core binding factor/acute myeloid leukemia factors contribute to the cell type-specific activity of the CD11a integrin gene promoter.

The CD11a/CD18 leukocyte integrin (LFA-1; also known as alphaL/beta2) mediates leukocyte transendothelial migration during immune and inflammatory responses and participates in lymphoma metastasis. CD11a/CD18 leukocyte-restricted expression is controlled by the CD11a gene promoter, which confers tissue-specific expression to reporter genes in vitro and in vivo. DNase I protection analysis of the CD11a proximal gene promoter revealed DNA-protein interactions centered at position -110 (CD11a-110).

Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.

Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution.

DNA-dependent protein kinase protects against heat-induced apoptosis.

Purified heat shock transcription factor 1 (HSF1) binds to both the regulatory and catalytic components of the DNA-dependent protein kinase (DNA-PK). This observation suggests that DNA-PK may have a physiological role in the heat shock response. To investigate this possibility, we performed a comparison of cell lines that were deficient in either the Ku protein or the DNA-PK catalytic subunit versus the same cell lines that had been rescued by the introduction of a functional gene.

Analysis of the phosphorylation of human heat shock transcription factor-1 by MAP kinase family members.

The activation of heat shock transcription factor-1 (HSF-1) after treatment of mammalian cells with stresses such as heat shock, heavy metals, or ethanol induces the synthesis of heat shock proteins. HSF-1 is phosphorylated at normal growth temperature and is hyperphosphorylated upon stress. We recently presented evidence that HSF-1 can be phosphorylated by the mitogen activated protein kinase, ERK1, and that such phosphorylation appears to negatively regulate the activity of HSF-1.

Heat shock transcription factor 1 binds selectively in vitro to Ku protein and the catalytic subunit of the DNA-dependent protein kinase.

Heat shock transcription factor 1 (HSF1) functions as the master regulator of the heat shock response in eukaryotes. We have previously shown that, in addition to its role as a transcription factor, HSF1 stimulates the activity of the DNA-dependent protein kinase (DNA-PK). DNA-PK is composed of two components: a 460-kDa catalytic subunit and a 70- and 86-kDa heterodimeric regulatory component, also known as the Ku protein.

Granulocyte-macrophage colony-stimulating factor, phorbol ester, and sodium butyrate induce the CD11c integrin gene promoter activity during myeloid cell differentiation.

To analyze the activity of the CD11c promoter during myeloid differentiation without the limitations of transient expression systems, we have stably transfected the myeloid U937 cell line with the pCD11C361-Luc plasmid, in which the expression of the firefly luciferase cDNA is driven by the CD11c promoter region -361/+43, previously shown to confer myeloid specificity to reporter genes. The stable transfectants (U937-C361) retained the ability to differentiate in response to phorbol-ester (PMA), sodium butyrate (SB), granulocyte-macrophage colony-stimulating factor (GM-CSF), and other differentiating agents. U937-C361 differentiation correlated with increased cellular luciferase levels, showing the inducibility of the CD11c promoter during myeloid differentiation and establishing the U937-C361 cells as a suitable system for studying the myeloid differentiation-inducing capacity of cytokines, growth, factors, and other biological response modifiers.

Hematopoietic cell-type-dependent regulation of leukocyte integrin functional activity: CD11b and CD11c expression inhibits LFA-1-dependent aggregation of differentiated U937 cells.

To study the influence of the cellular environment on the functional activity of leukocyte integrins and to analyze their involvement in hematopoietic cell differentiation, we have developed stable transfectants of LFA-1, Mac-1, and p150,95 (CD11a-c/CD18) leukocyte integrins in cultured cell lines whose differentiation can be induced in vitro. As on circulating leukocytes, the integrins expressed on U937 or K562 cells were expressed in a constitutively inactive state, as demonstrated by the lack of adhesion to their cellular counterreceptors or soluble ligands, the absence of CD18-dependent intercellular aggregation, and their inability to mediate adhesion to protein-coated plates. However, while leukocyte integrin adhesive functions in U937 cells were induced upon treatment with cellular agonists (e.

Regulation of expression of the LFA-1 and p150,95 leukocyte integrins: involvement of the CD11a and CD11c gene promoters.

Human Lymphocyte Associated Antigen-1 (LFA-1, CD11a/CD18, alpha L/beta 2) and p150,95 (CD11c/CD18, alpha X/beta 2) are cell surface alpha/beta heterodimers that, together with Mac-1 (CD11b/CD18, alpha M/beta 2) comprise the leukocyte-restricted beta 2 subfamily of integrins. LFA-1 is the only integrin expressed on all leukocyte lineages while p150,95 is exclusively expressed on cells of the myeloid lineage and on activated B lymphocytes and natural killer cells. The expression of the leukocyte integrins is regulated during cell activation and differentiation by transcriptional mechanisms.