Discovery of 9-Cyclopropylethynyl-2-(()-1-[1,4]dioxan-2-ylmethoxy)-6,7-dihydropyrimido[6,1-]isoquinolin-4-one (GLPG1205), a Unique GPR84 Negative Allosteric Modulator Undergoing Evaluation in a Phase II Clinical Trial.

GPR84 is a medium chain free fatty acid-binding G-protein-coupled receptor associated with inflammatory and fibrotic diseases. As the only reported antagonist of GPR84 (PBI-4050) that displays relatively low potency and selectivity, a clear need exists for an improved modulator. Structural optimization of GPR84 antagonist hit , identified through high-throughput screening, led to the identification of potent and selective GPR84 inhibitor GLPG1205 ().

Identification of GLPG/ABBV-2737, a Novel Class of Corrector, Which Exerts Functional Synergy With Other CFTR Modulators.

The deletion of phenylalanine at position 508 (F508del) in cystic fibrosis transmembrane conductance regulator (CFTR) causes a severe defect in folding and trafficking of the chloride channel resulting in its absence at the plasma membrane of epithelial cells leading to cystic fibrosis. Progress in the understanding of the disease increased over the past decades and led to the awareness that combinations of mechanistically different CFTR modulators are required to obtain meaningful clinical benefit. Today, there remains an unmet need for identification and development of more effective CFTR modulator combinations to improve existing therapies for patients carrying the F508del mutation.

Identification and Characterization of Novel CFTR Potentiators.

There is still a high unmet need for the treatment of most patients with cystic fibrosis (CF). The identification and development of new Cystic Fibrosis Transmembrane conductance Regulator (CFTR) modulators is necessary to achieve higher clinical benefit in patients. In this report we describe the characterization of novel potentiators.

Discovery of N-(3-Carbamoyl-5,5,7,7-tetramethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-lH-pyrazole-5-carboxamide (GLPG1837), a Novel Potentiator Which Can Open Class III Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels to a High Extent.

Cystic fibrosis (CF) is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). With the discovery of Ivacaftor and Orkambi, it has been shown that CFTR function can be partially restored by administering one or more small molecules. These molecules aim at either enhancing the amount of CFTR on the cell surface (correctors) or at improving the gating function of the CFTR channel (potentiators).

Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: from hit to clinic.

FFA2, also called GPR43, is a G-protein coupled receptor for short chain fatty acids which is involved in the mediation of inflammatory responses. A class of azetidines was developed as potent FFA2 antagonists. Multiparametric optimization of early hits with moderate potency and suboptimal ADME properties led to the identification of several compounds with nanomolar potency on the receptor combined with excellent pharmacokinetic (PK) parameters.

Triazolopyridines as selective JAK1 inhibitors: from hit identification to GLPG0634.

Janus kinases (JAK1, JAK2, JAK3, and TYK2) are involved in the signaling of multiple cytokines important in cellular function. Blockade of the JAK-STAT pathway with a small molecule has been shown to provide therapeutic immunomodulation. Having identified JAK1 as a possible new target for arthritis at Galapagos, the compound library was screened against JAK1, resulting in the identification of a triazolopyridine-based series of inhibitors represented by 3.

Preclinical characterization of GLPG0634, a selective inhibitor of JAK1, for the treatment of inflammatory diseases.

The JAKs receive continued interest as therapeutic targets for autoimmune, inflammatory, and oncological diseases. JAKs play critical roles in the development and biology of the hematopoietic system, as evidenced by mouse and human genetics. JAK1 is critical for the signal transduction of many type I and type II inflammatory cytokine receptors.

Essential validation of gene trap mouse ES cell lines: a test case with the gene Ttrap.

Gene trapping in mouse embryonic stem (ES) cells enables near-saturation vector-based insertional mutagenesis across the genome of this model organism. About 135,000 trapped ES cell lines are made available to the scientific community by the International Gene Trap Consortium (IGTC; www.genetrap.

Ttrap is an essential modulator of Smad3-dependent Nodal signaling during zebrafish gastrulation and left-right axis determination.

During vertebrate development, signaling by the TGFbeta ligand Nodal is critical for mesoderm formation, correct positioning of the anterior-posterior axis, normal anterior and midline patterning, and left-right asymmetric development of the heart and viscera. Stimulation of Alk4/EGF-CFC receptor complexes by Nodal activates Smad2/3, leading to left-sided expression of target genes that promote asymmetric placement of certain internal organs. We identified Ttrap as a novel Alk4- and Smad3-interacting protein that controls gastrulation movements and left-right axis determination in zebrafish.

Activation of NF-kappaB by Akt upregulates Snail expression and induces epithelium mesenchyme transition.

Carcinoma progression is associated with the loss of epithelial features, and the acquisition of mesenchymal characteristics and invasive properties by tumour cells. The loss of cell-cell contacts may be the first step of the epithelium mesenchyme transition (EMT) and involves the functional inactivation of the cell-cell adhesion molecule E-cadherin. Repression of E-cadherin expression by the transcription factor Snail is a central event during the loss of epithelial phenotype.

Neural crest-specific removal of Zfhx1b in mouse leads to a wide range of neurocristopathies reminiscent of Mowat-Wilson syndrome.

Mowat-Wilson syndrome is a recently delineated autosomal dominant developmental anomaly, whereby heterozygous mutations in the ZFHX1B gene cause mental retardation, delayed motor development, epilepsy and a wide spectrum of clinically heterogeneous features, suggestive of neurocristopathies at the cephalic, cardiac and vagal levels. However, our understanding of the etiology of this condition at the cellular level remains vague. This study presents the Zfhx1b protein expression domain in mouse embryos and correlates this with a novel mouse model involving a conditional mutation in the Zfhx1b gene in neural crest precursor cells.

Cloning and expression of the cyclophilin Bet v 7, and analysis of immunological cross-reactivity among the cyclophilin A family.

Allergic symptoms in sensitized individuals are caused by proteins named allergens. We report here the cloning and the production of the cyclophilin Bet v 7, one of the birch pollen allergens. Recombinant Bet v 7 was produced in bacteria and used to raise a rabbit anti-Bet v 7 antiserum.

Smicl is a novel Smad interacting protein and cleavage and polyadenylation specificity factor associated protein.

Ligand-bound receptors of the Transforming Growth Factor-beta (TGF-beta) family promote the formation of complexes between Smad proteins that subsequently accumulate in the nucleus and interact there with other transcriptional regulators, leading to modulation of target gene expression. We identified a novel nuclear protein, Smicl, which binds to Smad proteins. Smicl and Smads cooperate and enhance TGF-beta mediated activation of a Smad-responsive reporter gene.

Organization of the mouse Zfhx1b gene encoding the two-handed zinc finger repressor Smad-interacting protein-1.

SIP1, a member of the deltaEF1 family of two-handed zinc finger transcriptional repressors, has been identified as a Smad-binding protein. Recently, mutations in the human SIP1 gene (ZFHX1B) have been implicated in Hirschsprung disease. Here we document extensively the structure and transcriptional pattern of the mouse SIP1 gene (Zfhx1b) and compare it to homologues from other species.

Interaction between Smad-interacting protein-1 and the corepressor C-terminal binding protein is dispensable for transcriptional repression of E-cadherin.

deltaEF1 and SIP1 (or Zfhx1a and Zfhx1b, respectively) are the only known members of the vertebrate Zfh1 family of homeodomain/zinc finger-containing proteins. Similar to other transcription factors, both Smad-interacting protein-1 (SIP1) and deltaEF1 are capable of repressing E-cadherin transcription through binding to the E2 boxes located in its promoter. In the case of deltaEF1, this repression has been proposed to occur via interaction with the corepressor C-terminal binding protein (CtBP).

Mice lacking ZFHX1B, the gene that codes for Smad-interacting protein-1, reveal a role for multiple neural crest cell defects in the etiology of Hirschsprung disease-mental retardation syndrome.

Recently, mutations in ZFHX1B, the gene that encodes Smad-interacting protein-1 (SIP1), were found to be implicated in the etiology of a dominant form of Hirschsprung disease-mental retardation syndrome in humans. To clarify the molecular mechanisms underlying the clinical features of SIP1 deficiency, we generated mice that bear a mutation comparable to those found in several human patients. Here, we show that Zfhx1b-knockout mice do not develop postotic vagal neural crest cells, the precursors of the enteric nervous system that is affected in patients with Hirschsprung disease, and they display a delamination arrest of cranial neural crest cells, which form the skeletomuscular elements of the vertebrate head.

Transforming growth factor beta signalling in vitro and in vivo: activin ligand-receptor interaction, Smad5 in vasculogenesis, and repression of target genes by the deltaEF1/ZEB-related SIP1 in the vertebrate embryo.

The identification and characterization of components of the transforming growth factor beta (TGFbeta) signalling pathway are proceeding at a very fast pace. To illustrate a number of our activities in this field, we first summarize our work aiming at the selection from a large collection of single residue substitution mutants of two activin A polypeptides in which D27 and K102, respectively, have been modified. This work has highlighted the importance of K102 and its positive charge for binding to activin type II receptors.

Correlation between ALK-6 (BMPR-IB) distribution and responsiveness to osteogenic protein-1 (BMP-7) in embryonic mouse bone rudiments.

Osteogenic protein-1 (OP-1) or bone morphogenetic protein-7 (BMP-7) stimulates cartilage formation in mouse bone rudiments in vitro but arrests terminal differentiation of prehypertrophic chondrocytes into hypertrophic chondrocytes. In this study we report that these effects of OP-1 depend on the developmental stage of the bone rudiment, early stages (E14 and E15 metatarsals) being most responsive. E17 metatarsals that already contained a hypertrophic area that had initiated mineralization were no longer affected by OP-1.

SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5'-CACCT sequences in candidate target genes.

Activation of transforming growth factor beta receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. We describe a novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast and in vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation.

A novel expression cloning method to isolate mammalian transcription factors in Schizosaccharomyces pombe.

We describe a novel expression cloning strategy in the fission yeast for the isolation of mammalian transcription factors using a mammalian promoter as target. This strategy is possible because of the conservation between mammalian cells and Schizosaccharomyces pombe of the mechanism that leads to the selection of the transcription start site. It also opens new perspectives to investigate the transcriptional regulation of genes for which detailed promoter analysis is difficult.

Identical cis-acting elements and related trans-acting factors control activity of nonviral promoter in Schizosaccharomyces pombe and mammalian cells.

We have analyzed the transcriptional activity of the human plasminogen activator inhibitor-1 promoter in the fission yeast Schizosaccharomyces pombe. This promoter is active in S. pombe, and the initiation site of transcription corresponds to the site identified previously in mammalian cells.

Identification of a multihormone responsive enhancer far upstream from the human tissue-type plasminogen activator gene.

A 2.4-kilobase (kb) DNA fragment, located 7.1 kb upstream from the human tissue-type plasminogen activator (t-PA) gene (t-PA2.