Transcriptional regulation and functional characterization of the oxysterol / EBI2 system in primary human macrophages

Oxysterols such as 7 alpha, 25-dihydoxycholesterol (7a,25-OHC) are natural ligands for the Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183), a G protein–coupled receptor (GPCR) highly expressed in immune cells and required for adaptive immune responses [7;10]. Activation of EBI2 by specific oxysterols leads to directed cell migration of B cells in lymphoid tissues. While the ligand gradient necessary for this critical process of the adapted immune response is established by a stromal cells subset [13], here we investigate the involvement of the oxysterol / EBI2 system in the innate immune response. First, we show that primary human macrophages express EBI2 and the enzymes necessary for ligand production such as cholesterol 25-hydroxylase (CH25H) and oxysterol 7alpha-hydroxylase (CYP7B1). Furthermore, challenge of monocyte-derived macrophages with lipopolysaccharide (LPS) triggers a strong continuous up-regulation of CH25H and CYP7B1 in comparison to a transient increase in EBI2 expression. Activation of EBI2 expressed on macrophages leads to release of intracellular calcium and to directed cell migration. Supernatants of LPS-stimulated macrophages show an autocrine activation of EBI2 indicating that an induction of CH25H and CYP7B1 results in an enhanced production and release of oxysterols into the cellular environment. This is the first study characterizing the oxysterol / EBI2 pathway in primary human cells. Given the crucial functional role of macrophages in the innate immune response these results encourage further exploration of a possible link to systemic autoimmunity

RIPK3-MLKL-Mediated Neutrophil Death Requires Concurrent Activation of Fibroblast Activation Protein-α.

Cytokine-primed neutrophils can undergo a nonapoptotic type of cell death using components of the necroptotic pathway, including receptor-interacting protein kinase-3 (RIPK3), mixed lineage kinase-like (MLKL) and NADPH oxidase. In this report, we provide evidence for a potential role of serine proteases in CD44-mediated necroptotic death of GM-CSF-primed human neutrophils. Specifically, we observed that several inhibitors known to block the enzymatic function of fibroblast activation protein-α (FAP-α) were able to block CD44-mediated reactive oxygen species production and cell death, but not FAS receptor-mediated apoptosis. To understand how FAP-α is involved in this nonapoptotic death pathway, we performed immunoblotting experiments in the presence and absence of inhibitors of RIPK3, MLKL, p38 MAPK, PI3K, and FAP-α. The results of these experiments suggested that FAP-α is active in parallel with RIPK3, MLKL, and p38 MAPK activation but proximal to PI3K and NADPH oxidase activation. Interestingly, neutrophils isolated from the joints of patients suffering from rheumatoid arthritis underwent a GM-CSF-independent necroptosis following CD44 ligation; this effect was also blocked by both FAP-α and MLKL inhibitors. Taken together, our evidence shows that the RIPK3-MLKL pathway activates NADPH oxidase but requires, in addition to p38 MAPK and PI3K, a serine protease activity, whereby FAP-α is the most likely candidate. Thus, FAP-α could be a potential drug target in neutrophilic inflammatory responses to avoid exaggerated nonapoptotic neutrophil death, leading to tissue damage

Identification and characterization of small molecule modulators of the EBI2 receptor

Oxysterols such as 7a, 25-dihydroxycholesterol (7a,25-OHC) have recently been identified as natural ligands for a G protein-coupled receptor called Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183) 1, 2. EBI2 is highly expressed in immune cells and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes 3. In order to further delineate the physiological role of the oxysterol/EBI2 pathway in health and disease, here we describe the isolation and characterization of a potent small molecule antagonist for the EBI2 receptor. Prior to the isolation of the natural ligand, we first identified a surrogate small molecule agonist NIBR51 (NVP-AIU198) 1, which enabled identification of inhibitors of receptor activation. One antagonist called NIBR127 (NVP-AQV967) 2 was used as a starting point for a medicinal chemistry campaign which yielded NIBR189 (NVP-CHY976) 4m. This compound was extensively characterized in binding and various functional signaling assays to show that it is a potent and selective antagonist for the EBI2 receptor. Furthermore we have used NIBR189 (NVP-CHY976) 4m to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells

Discovery of dihydroisoquinolinone derivatives as novel inhibitors of the p53-MDM2 interaction with a distinct binding mode

Blocking the interaction between the p53 tumor suppressor and its regulatory protein MDM2 is a promising therapeutic concept under current investigation in oncology drug research. We report here the discovery of the first representatives of a new class of small molecule inhibitors of this protein-protein interaction: the dihydroisoquinolinones. Starting from an initial hit identified by virtual screening, a derivatization program has resulted in compound 11, a low nanomolar inhibitor of the p53-MDM2 interaction showing significant cellular activity. Initially based on a binding mode hypothesis, this effort was then guided by a X-ray co-crystal structure of MDM2 in complex with one of the synthesized analogues. The X-ray structure revealed an unprecedented binding mode for p53-MDM2 inhibitors

Design of potent and selective covalent inhibitors of Bruton’s Tyrosine Kinase targeting an inactive conformation

Bruton’s tyrosine kinase (BTK) is a member of the TEC kinase family and is selectively expressed in a subset of immune cells. It is a key regulator of antigen receptor signaling in B cells and of Fc receptor signaling in mast cells and macrophages. A BTK inhibitor will likely have a positive impact on autoimmune diseases which are caused by autoreactive B cells and immune-complex driven inflammation. We report the design, optimization, and characterization of potent and selective covalent BTK inhibitors. Starting from the selective reversible inhibitor 3 binding to an inactive conformation of BTK, we designed covalent irreversible compounds by attaching an electrophilic warhead to reach Cys481. The first prototype 4 covalently modified BTK and showed an excellent kinase selectivity including several Cys-containing kinases, validating the design concept. In addition, this compound blocked FcγR-mediated hypersensitivity in vivo. Optimization of whole blood potency and metabolic stability resulted in compounds such as 8, which maintained the excellent kinase selectivity and showed improved BTK occupancy in vivo

Discovery of C-(1-aryl-cyclohexyl)-methylamines as selective, orally available inhibitors of dipeptidyl peptidase IV.

The successful launches of dipeptidyl peptidase IV (DPP IV) inhibitors as oral anti-diabetics warrant and spur the further quest for additional chemical entities in this promising class of therapeutics. Numerous pharmaceutical companies have pursued their proprietary candidates towards the clinic, resulting in a large body of published chemical structures associated with DPP IV. Herein, we report the discovery of a novel chemotype for DPPIV inhibition based on the C-(1-aryl-cyclohexyl)-methylamine scaffold and its optimization to compounds which selectively inhibit DPP IV at low-nM potency and exhibit an excellent oral phamacokinetic profile in the rat

Structural states of Hdm2 and HdmX: X-ray elucidation of adaptations and binding interactions for different chemical compound classes

Hdm2 (human MDM2) counteracts p53 function by direct binding to p53 and by ubiquitin-dependent p53 protein degradation. Activation of p53 by inhibitors of the p53-Hdm2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. In addition, HdmX (human MDMX, human MDM4) was also identified as an important therapeutic target to efficiently reactivate p53, and it is likely that dual inhibition of Hdm2 and HdmX is beneficial. Here, we report four new X-ray structures for Hdm2 and five new X-ray structures for HdmX complexes, involving different classes of synthetic compounds. We also reveal the key additive 18-crown-ether, which we have discovered to enable HdmX crystallization and show its stabilization of various Lys-residues. In addition, we report the previously unpublished details of X-ray structure determinations for eight further Hdm2 complexes, including the clinical trial compounds NVP-CGM097 and NVP-HDM201. An analysis of all compound binding modes reveals new and deepened insights into the possible adaptations and structural states of Hdm2 (e.g. flip of F55; flip of Y67; reorientation of H96) and HdmX (e.g. flip of H55; dimer induction), enabling key binding interactions for different compound classes. In order to make comparisons easier, we have used the same numbering for Hdm2 and HdmX. Taken together, these structural insights should prove useful for the design and optimization of further selective and/or dual Hdm2/HdmX inhibitors

Discovery of LOU064 (Remibrutinib), a Potent and Highly Selective Covalent Inhibitor of Bruton’s Tyrosine Kinase

Bruton’s tyrosine kinase (BTK), a cytoplasmic tyrosine kinase, plays a central role in immunity and is considered an attractive target for treating autoimmune diseases. The use of currently marketed covalent BTK inhibitors is limited to oncology indications based on their suboptimal kinase selectivity. We describe the discovery and preclinical profile of LOU064 (25), a potent, highly selective covalent BTK inhibitor. LOU064 exhibits an exquisite kinase selectivity due to binding to an inactive conformation of BTK and has the potential for a best-in-class covalent BTK inhibitor for the treatment of autoimmune diseases. It demonstrates potent in vivo target occupancy with an EC90 of 1.6 mg/kg and dose-dependent efficacy in rat collagen-induced arthritis. LOU064 is currently being tested in Phase 2 clinical studies for chronic spontaneous urticaria and Sjoegren’s Syndrome

Identification and Characterization of Small Molecule Modulators of the Epstein–Barr Virus-Induced Gene 2 (EBI2) Receptor

Oxysterols have recently been identified as natural ligands for a G protein-coupled receptor called EBI2 (aka GPR183) (Nature 2011, 475, 524; 519). EBI2 is highly expressed in immune cells (J. Biol. Chem. 2006, 281, 13199), and its activation has been shown to be critical for the adaptive immune response and has been genetically linked to autoimmune diseases such as type I diabetes (Nature 2010, 467, 460). Here we describe the isolation of a potent small molecule antagonist for the EBI2 receptor. First, we identified a small molecule agonist NIBR51 (<b>1</b>), which enabled identification of inhibitors of receptor activation. One antagonist called NIBR127 (<b>2</b>) was used as a starting point for a medicinal chemistry campaign, which yielded NIBR189 (<b>4m</b>). This compound was extensively characterized in binding and various functional signaling assays. Furthermore, we have used <b>4m</b> to block migration of a monocyte cell line called U937, suggesting a functional role of the oxysterol/EBI2 pathway in these immune cells