Luc Nelles

The C-terminal domain of Mad-like signal transducers is sufficient for biological activity in the Xenopus embryo and transcriptional activation

We report the characterization of two vertebrate homologs of Drosophila mothers against dpp (Mad) isolated from the mouse and the Xenopus embryo, named MusMLP (mad-like protein) and XenMLP, respectively, together with a summary of their expression patterns in the embryo. Overexpression of XenMLP causes ventralization of Xenopus embryos and we demonstrate that the C-terminal domain is necessary and sufficient to confer this biological effect. This domain also has the potential for transcriptional activation, as shown in one-hybrid assays in mammalian cells. We further demonstrate that MLPs are multidomain proteins by showing a cis-negative effect of the N-terminal domain on the transactivation by the C-terminal domain and that the proline-rich, middle domain maximizes the activity of the C-terminal domain. We also mapped the MusMLP gene to a region on mouse chromosome 13 that corresponds to a region on human chromosome 5q that contains cancer-related genes.

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. The most advanced compound, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.

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

During vertebrate development, signaling by the TGFβ 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. Morpholino-mediated Ttrap knockdown increases Smad3 activity, leading to ectopic expression of snail1a and apparent repression of e-cadherin, thereby perturbing cell movements during convergent extension, epiboly and node formation. Thus, although the role of Smad proteins in mediating Nodal signaling is well-documented, the functional characterization of Ttrap provides insight into a novel Smad partner that plays an essential role in the fine-tuning of this signal transduction cascade.

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. Optimization within this chemical series led to identification of GLPG0634 (65, filgotinib), a selective JAK1 inhibitor currently in phase 2B development for RA and phase 2A development for Crohns disease (CD).

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 pre-hypertrophic 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. We then investigated whether the sensitivity of the early long bone rudiments to OP-1 correlated with high expression of the OP-1 binding type I serine/threonine kinase receptors (activin receptor-like kinase: ALK-2/ActR-I, ALK-3/BMPR-IA or ALK-6/BMPR-IB) at this early stage. We did not find any significant difference in overall mRNA levels of these ALKs between stages E14 through E17 as assessed by RNase protection assays. However, by immunohistochemistry we found that ALK-6 staining was strong in E14 early cartilage primordium and its future perichondrium but dropped sharply to low levels in these cell types until onset of chondrocyte (pre)hypertrophy at E16. By contrast, ALK-2 and ALK-3 immunostainings in E14 were barely detectable. We also examined by immunohistochemistry the local synthesis of OP-1. OP-1 was present in E14 early chondrocytes and forming perichondrium but in low amounts; however, production of OP-1 increased in these cell types with age. All three receptor types as well as OP-1 were present in significant amounts in prehypertrophic chondrocytes and late hypertrophic chondrocytes including those undergoing mineralization. The temporary high immunostaining for ALK-6 in the early proliferating chondrocytes and future perichondrium of E14 bone rudiments, and its absence in older bones correlated with the sensitivity of chondrocytes and perichondrium to (exogenous) OP-1. We therefore propose that the effects of OP-1 on these cells in vitro are mediated by ALK-6/BMPR-IB. We furthermore conclude that locally produced OP-1 is a potential autocrine/paracrine growth factor. Increased local production of OP-1 may be partially responsible for the age-related decrease in responsiveness to exogenous OP-1 with respect to hypertrophy and mineralization of cartilage.

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). Here we describe the discovery of a novel potentiator GLPG1837, which shows enhanced efficacy on CFTR mutants harboring class III mutations compared to Ivacaftor, the first marketed potentiator. The optimization of potency, efficacy, and pharmacokinetic profile will be described.