Publications by authors named "Andreas Billich"

73 Publications

Structure-Based and Property-Driven Optimization of -Aryl Imidazoles toward Potent and Selective Oral RORγt Inhibitors.

J Med Chem 2019 12 3;62(23):10816-10832. Epub 2019 Dec 3.

Global Discovery Chemistry , 181 Massachusetts Avenue , 02139 Cambridge , Massachusetts , United States.

Retinoic acid receptor-related orphan receptor gamma-t (RORγt) is considered to be the master transcription factor for the development of Th17 cells that produce proinflammatory cytokines such as IL-17A. Overproportionate Th17 cell abundance is associated with the pathogenesis of many inflammatory conditions including psoriasis. In a high-throughput fluorescence resonance energy transfer (FRET) screen, we identified compound 1 as a hit with promising lipophilic efficiency (LipE). Using structure-based drug design based on a number of X-ray cocrystal structures, we morphed this hit class into potent imidazoles, exemplified by compound . To improve the poor absorption, distribution, metabolism, and excretion (ADME) properties of neutral imidazoles, we extended our ligands with carboxylic acid substituents toward a polar, water-rich area of the protein. This highly lipophilicity-efficient modification ultimately led to the discovery of compound , a potent and selective inhibitor of RORγt with good ADME properties and excellent in vivo pharmacokinetics. This compound showed good efficacy in an in vivo delayed-type hypersensitivity pharmacology model in rats.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01291DOI Listing
December 2019

Optimizing a Weakly Binding Fragment into a Potent RORγt Inverse Agonist with Efficacy in an in Vivo Inflammation Model.

J Med Chem 2018 08 24;61(15):6724-6735. Epub 2018 Jul 24.

The transcription factor RORγt is an attractive drug-target due to its role in the differentiation of IL-17 producing Th17 cells that play a critical role in the etiopathology of several autoimmune diseases. Identification of starting points for RORγt inverse agonists with good properties has been a challenge. We report the identification of a fragment hit and its conversion into a potent inverse agonist through fragment optimization, growing and merging efforts. Further analysis of the binding mode revealed that inverse agonism was achieved by an unusual mechanism. In contrast to other reported inverse agonists, there is no direct interaction or displacement of helix 12 observed in the crystal structure. Nevertheless, compound 9 proved to be efficacious in a delayed-type hypersensitivity (DTH) inflammation model in rats.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00529DOI Listing
August 2018

Interferon regulatory factor 5 and nuclear factor kappa-B exhibit cooperating but also divergent roles in the regulation of pro-inflammatory cytokines important for the development of TH1 and TH17 responses.

FEBS J 2018 08 11;285(16):3097-3113. Epub 2018 Jul 11.

Department of Autoimmunity, Transplantation & Inflammation, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland.

A large body of data demonstrates that interferon regulatory factor 5 (IRF5) and nuclear factor kappa B (NF-κB) are the two major transcription factors in classically activated macrophages responsible for the transcriptional control of proinflammatory genes. Although recent evidence suggests that IRF5 interacts with certain members of the nuclear factor kappa B pathway, the extent of cooperation and its implications in disease are ambiguous. Since both pathways are known for their strong contributions in TLR8 signaling we used the human monocytic cell line THP-1.Dual, featuring gene reporters for NF-κB and IRFs, to simultaneously study the roles of IRF5 and the NF-κB subunit p65 in TLR8-mediated gene reporter activities. Furthermore, we profiled from these cells the proinflammatory cytokines involved in the differentiation of TH1 and TH17 cells. After ablation of IRF5 and/or p65 we activated the resultant cells with the TLR8 agonists R848 or the psoriasis-associated antimicrobial peptide LL-37 complexed with ssRNA and demonstrate that IRF5 deficiency drastically impairs the secretion of IL-1β, IL-6, IL-12, IL-23 and TNFα. In contrast, the lack of p65 impaired only IL-6, IL-12, and IL-23 secretion. Furthermore, we discovered that upon TLR8 stimulation, IRF5 but not NF-κB signaling is essential to provide a cytokine milieu supporting TH1 responses. Additionally, we demonstrate that IRF5 and NF-κB cooperate to provide a cytokine milieu supporting TH17 responses. Therefore, the distinct role of IRF5 in the intricate signaling network downstream of TLR8 may open new treatment options interfering with but not disrupting NF-κB signaling in human diseases.
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http://dx.doi.org/10.1111/febs.14600DOI Listing
August 2018

Pharmacological inhibition of RORγt suppresses the Th17 pathway and alleviates arthritis in vivo.

PLoS One 2017 20;12(11):e0188391. Epub 2017 Nov 20.

Autoimmunity, Transplantation and Inflammation, Novartis Institutes for BioMedical Research, Basel, Switzerland.

Retinoic acid receptor-related-orphan-receptor-C (RORγt) is the key transcription factor that is driving the differentiation of IL-17 producing T-helper 17 (Th17) cells that are implicated in the pathology of various autoimmune and inflammatory diseases. Based on the importance of RORγt in promoting Th17-driven pathology, there is considerable interest to develop low-molecular-weight compounds with the aim of inhibiting the transcriptional activity of this nuclear hormone receptor. In this article, we describe the in vitro and in vivo pharmacology of a potent and selective small-molecular-weight RORγt inverse agonist. The compound binds to the ligand binding domain (LBD) of RORγt leading to displacement of a co-activator peptide. We show for the first time that a RORγt inverse agonist down-regulates permissive histone H3 acetylation and methylation at the IL17A and IL23R promoter regions, thereby providing insight into the transcriptional inhibition of RORγt-dependent genes. Consistent with this, the compound effectively reduced IL-17A production by polarized human T-cells and γδT-cells and attenuated transcription of RORγt target genes. The inhibitor showed good in vivo efficacy in an antigen-induced arthritis model in rats and reduced the frequencies of IL-17A producing cells in ex vivo recall assays. In summary, we demonstrate that inhibiting RORγt by a low-molecular-weight inhibitor results in efficient and selective blockade of the pro-inflammatory Th17/IL-17A pathway making it an attractive target for Th17-mediated disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188391PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695821PMC
December 2017

Feasibility and physiological relevance of designing highly potent aminopeptidase-sparing leukotriene A4 hydrolase inhibitors.

Sci Rep 2017 10 19;7(1):13591. Epub 2017 Oct 19.

Autoimmunity, Transplantation and Inflammation, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

Leukotriene A4 Hydrolase (LTA4H) is a bifunctional zinc metalloenzyme that comprises both epoxide hydrolase and aminopeptidase activity, exerted by two overlapping catalytic sites. The epoxide hydrolase function of the enzyme catalyzes the biosynthesis of the pro-inflammatory lipid mediator leukotriene (LT) B4. Recent literature suggests that the aminopeptidase function of LTA4H is responsible for degradation of the tripeptide Pro-Gly-Pro (PGP) for which neutrophil chemotactic activity has been postulated. It has been speculated that the design of epoxide hydrolase selective LTA4H inhibitors that spare the aminopeptidase pocket may therefore lead to more efficacious anti-inflammatory drugs. In this study, we conducted a high throughput screen (HTS) for LTA4H inhibitors and attempted to rationally design compounds that would spare the PGP degrading function. While we were able to identify compounds with preference for the epoxide hydrolase function, absolute selectivity was not achievable for highly potent compounds. In order to assess the relevance of designing such aminopeptidase-sparing LTA4H inhibitors, we studied the role of PGP in inducing inflammation in different settings in wild type and LTA4H deficient (LTA4H KO) animals but could not confirm its chemotactic potential.  Attempting to design highly potent epoxide hydrolase selective LTA4H inhibitors, therefore seems to be neither feasible nor relevant.
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http://dx.doi.org/10.1038/s41598-017-13490-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5648829PMC
October 2017

Retinoic-acid-orphan-receptor-C inhibition suppresses Th17 cells and induces thymic aberrations.

JCI Insight 2017 03 9;2(5):e91127. Epub 2017 Mar 9.

Preclinical Safety, Novartis Institutes for BioMedical Research, Basel, Switzerland.

Retinoic-acid-orphan-receptor-C (RORC) is a master regulator of Th17 cells, which are pathogenic in several autoimmune diseases. Genetic deficiency in mice, while preventing autoimmunity, causes early lethality due to metastatic thymic T cell lymphomas. We sought to determine whether pharmacological RORC inhibition could be an effective and safe therapy for autoimmune diseases by evaluating its effects on Th17 cell functions and intrathymic T cell development. RORC inhibitors effectively inhibited Th17 differentiation and IL-17A production, and delayed-type hypersensitivity reactions. In vitro, RORC inhibitors induced apoptosis, as well as and mRNA downregulation, in mouse and nonhuman primate thymocytes, respectively. Chronic, 13-week RORC inhibitor treatment in rats caused progressive thymic alterations in all analyzed rats similar to those in -deficient mice prior to T cell lymphoma development. One rat developed thymic cortical hyperplasia with preneoplastic features, including increased mitosis and reduced IKAROS expression, albeit without skewed T cell clonality. In summary, pharmacological inhibition of RORC not only blocks Th17 cell development and related cytokine production, but also recapitulates thymic aberrations seen in -deficient mice. While RORC inhibition may offer an effective therapeutic principle for Th17-mediated diseases, T cell lymphoma with chronic therapy remains an apparent risk.
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http://dx.doi.org/10.1172/jci.insight.91127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333964PMC
March 2017

Synthesis and Biological Evaluation of New Triazolo- and Imidazolopyridine RORγt Inverse Agonists.

ChemMedChem 2016 12 30;11(24):2640-2648. Epub 2016 Nov 30.

Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Campus, 4002, Basel, Switzerland.

Retinoic-acid-related orphan receptor γt (RORγt) is a key transcription factor implicated in the production of pro-inflammatory Th17 cytokines, which drive a number of autoimmune diseases. Despite diverse chemical series having been reported, combining high potency with a good physicochemical profile has been a very challenging task in the RORγt inhibitor field. Based on available chemical structures and incorporating in-house knowledge, a new series of triazolo- and imidazopyridine RORγt inverse agonists was designed. In addition, replacement of the terminal cyclopentylamide metabolic soft spot by five-membered heterocycles was investigated. From our efforts, we identified an optimal 6,7,8-substituted imidazo[1,2-a]pyridine core system and a 5-tert-butyl-1,2,4-oxadiazole as cyclopentylamide replacement leading to compounds 10 ((S)-N-(8-((4-(cyclopentanecarbonyl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide) and 33 ((S)-N-(8-((4-(5-(tert-butyl)-1,2,4-oxadiazol-3-yl)-3-methylpiperazin-1-yl)methyl)-7-methylimidazo[1,2-a]pyridin-6-yl)-2-methylpyrimidine-5-carboxamide). Both derivatives showed good pharmacological potencies in biochemical and cell-based assays combined with excellent physicochemical properties, including low to medium plasma protein binding across species. Finally, 10 and 33 were shown to be active in a rodent pharmacokinetic/pharmacodynamic (PK/PD) model after oral gavage at 15 mg kg , lowering IL-17 cytokine production in ex vivo antigen recall assays.
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http://dx.doi.org/10.1002/cmdc.201600500DOI Listing
December 2016

Sphingosine 1-Phosphate Produced by Sphingosine Kinase 2 Intrinsically Controls Platelet Aggregation In Vitro and In Vivo.

Circ Res 2015 Jul 30;117(4):376-87. Epub 2015 Jun 30.

From the Medizinische Klinik und Poliklinik I, Klinikum der Universität München (N.U., F.G., M.-L.v.B., S.C., F.R., M.O., V.B., J.B., I.S., M.L., K.R.L., S.M.), Department of Applied Physics, Center for NanoSciences (K.R.L.), and Walther-Straub-Institute of Pharmacology and Toxicology (M.M.y.S.), Ludwig-Maximilians-Universität, Munich, Germany; DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance, Munich, Germany (N.U., F.G., M.-L.v.B., S.C., F.R., M.O., J.B., I.S., M.L., M.M.y.S., S.M.); Heart Failure Institute, Research Center for Translational Medicine and Department of Cardiovascular Medicine, East Hospital, Tongji University School of Medicine, Shanghai, China (L.Z.); Institute of Pharmacology, University of Bern, Bern, Switzerland (A.H.); Pharmazentrum Frankfurt/ZAFES, Goethe University Hospital, Frankfurt am Main, Germany (J.M.P.); and Preclinical Safety (D.L., E.P.), and Autoimmunity, Transplantation and Inflammation (C.B., A.B., T.B.), Novartis Institutes for BioMedical Research, Basel, Switzerland.

Rationale: Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function.

Objective: To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function.

Methods And Results: We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo.

Conclusions: We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis.
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http://dx.doi.org/10.1161/CIRCRESAHA.115.306901DOI Listing
July 2015

Reduced Activity of Sphingosine-1-Phosphate Lyase Induces Podocyte-related Glomerular Proteinuria, Skin Irritation, and Platelet Activation.

Toxicol Pathol 2015 Jul 27;43(5):694-703. Epub 2015 Jan 27.

Preclinical Safety, Novartis Institutes for BioMedical Research, Basel, Switzerland.

Sphingosine-1-phosphate (S1P) lyase is considered as a drug target in autoimmune diseases based on the protective effect of reducing activity of the enzyme in animal models of inflammation. Since S1P lyase deficiency in mice causes a severe, lethal phenotype, it was of interest to investigate any pathological alterations associated with only partially reduced activity of S1P lyase as may be encountered upon pharmacological inhibition. Both genetic reduction of S1P lyase activity in mice and inhibition of S1P lyase with a low-molecular-weight compound in rats consistently resulted in podocyte-based kidney toxicity, which is the most severe finding. In addition, skin irritation and platelet activation were observed in both instances. The similarity of the findings in both the genetic model and the pharmacological study supports the value of analyzing inducible partially target-deficient mice for safety assessment. If the findings described in rodents translate to humans, target-related toxicity, particularly podocyte dysfunction, may limit chronic systemic treatment of autoimmune diseases with S1P lyase inhibitors. Furthermore, partial deficiency or inhibition of S1P lyase appears to provide an in vivo rodent model to enable studies on the mechanism of podocyte dysfunction.
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http://dx.doi.org/10.1177/0192623314565650DOI Listing
July 2015

Orally active 7-substituted (4-benzylphthalazin-1-yl)-2-methylpiperazin-1-yl]nicotinonitriles as active-site inhibitors of sphingosine 1-phosphate lyase for the treatment of multiple sclerosis.

J Med Chem 2014 Jun 21;57(12):5074-84. Epub 2014 May 21.

Novartis Institutes for BioMedical Research , Basel, CH-4002, Switzerland.

Sphingosine 1-phosphate (S1P) lyase has recently been implicated as a therapeutic target for the treatment of multiple sclerosis (MS), based on studies in a genetic mouse model. Potent active site directed inhibitors of the enzyme are not known so far. Here we describe the discovery of (4-benzylphthalazin-1-yl)-2-methylpiperazin-1-yl]nicotinonitrile 5 in a high-throughput screen using a biochemical assay, and its further optimization. This class of compounds was found to inhibit catalytic activity of S1PL by binding to the active site of the enzyme, as seen in the cocrystal structure of derivative 31 with the homodimeric human S1P lyase. 31 induces profound reduction of peripheral T cell numbers after oral dosage and confers pronounced protection in a rat model of multiple sclerosis. In conclusion, this novel class of direct S1P lyase inhibitors provides excellent tools to further explore the therapeutic potential of T cell-targeted therapies in multiple sclerosis and other autoimmune and inflammatory diseases.
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http://dx.doi.org/10.1021/jm500338nDOI Listing
June 2014

Second generation S1P pathway modulators: research strategies and clinical developments.

Biochim Biophys Acta 2014 May 12;1841(5):745-58. Epub 2013 Nov 12.

Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland. Electronic address:

Multiple Sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 2010, fingolimod/FTY720 (Gilenya, Novartis) was approved as the first oral treatment for relapsing forms of MS. Fingolimod causes down-modulation of S1P1 receptors on lymphocytes which prevents the invasion of autoaggressive T cells into the CNS. In astrocytes, down-modulation of S1P1 by the drug reduces astrogliosis, a hallmark of MS, thereby allowing restoration of productive astrocyte communication with other neural cells and the blood brain barrier. Animal data further suggest that the drug directly supports the recovery of nerve conduction and remyelination. In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by the drug. Fingolimod binds to 4 of the 5 known S1P receptor subtypes, and significant efforts were made over the past 5 years to develop next generation S1P receptor modulators and determine the minimal receptor selectivity needed for maximal therapeutic efficacy in MS patients. Other approaches considered were competitive antagonists of the S1P1 receptor, inhibitors of the S1P lyase to prevent S1P degradation, and anti-S1P antibodies. Below we discuss the current status of the field, and the functional properties of the most advanced compounds. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.
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http://dx.doi.org/10.1016/j.bbalip.2013.11.001DOI Listing
May 2014

PIKfyve, a class III PI kinase, is the target of the small molecular IL-12/IL-23 inhibitor apilimod and a player in Toll-like receptor signaling.

Chem Biol 2013 Jul;20(7):912-21

Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA.

Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohn's disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling.
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http://dx.doi.org/10.1016/j.chembiol.2013.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878021PMC
July 2013

Partial deficiency of sphingosine-1-phosphate lyase confers protection in experimental autoimmune encephalomyelitis.

PLoS One 2013 27;8(3):e59630. Epub 2013 Mar 27.

Novartis Institutes for BioMedical Research, Basel, Switzerland.

Background: Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span.

Methodology: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation.

Principal Findings: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS.

Significance: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0059630PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3609791PMC
September 2013

Assay to measure the secretion of sphingosine-1-phosphate from cells induced by S1P lyase inhibitors.

Biochem Biophys Res Commun 2013 Apr 14;433(3):345-8. Epub 2013 Mar 14.

Novartis Institutes for BioMedical Research, Basel, Switzerland.

Inhibitors of the sphingosine-1-phosphate (S1P) degrading enzyme S1P lyase (SPL) may be useful in the therapy of inflammatory diseases by preventing lymphocyte recruitment to diseased tissues. Here we describe a cellular assay for such inhibitors, which takes advantage of the observation that a fraction of the intracellular S1P accumulated in the presence of SPL inhibitors is secreted into the medium of cultured cells. The secreted S1P is then quantified using an S1P-sensitive reporter cell line. In the routine assay protocol, human HEK293T cells are treated with SPL inhibitors in the presence of phosphatase inhibitors and sphingosine; while the phosphatase inhibitors are included to prevent the degradation of S1P secreted from the cells, sphingosine is added as source for intracellular S1P that is prone to SPL degradation. The secreted S1P in the supernatant of the cell cultures is then quantified by measuring calcium flux induced in CHO-K1 cells expressing the human S1P3 receptor. Using this method SPL inhibitors were shown to induce a concentration-dependent increase of extracellular S1P under the conditions used; thus, the assay allows for the ranking of SPL inhibitors according to their potency on living cells.
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http://dx.doi.org/10.1016/j.bbrc.2013.03.004DOI Listing
April 2013

Cellular assay for the characterization of sphingosine-1-phosphate lyase inhibitors.

Anal Biochem 2013 Mar 13;434(2):247-53. Epub 2012 Dec 13.

Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland.

Sphingosine-1-phosphate (S1P) lyase represents a target for therapeutic intervention in immune regulation. Inhibitors of the lyase can be identified by established biochemical assays, but a cellular test system for such inhibitors has not been described so far. We found that silencing or inhibition of S1P lyase with short interfering RNA (siRNA) or active site-directed inhibitors in cultured mammalian cells does not cause a relevant increase of S1P in the cells as measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, the addition of sphingosine to cultures of cell lines or primary cells provides a source of intracellular S1P that is susceptible to degradation by the lyase and, hence, increases on inhibition or silencing of the enzyme. The assay was optimized with respect to sphingosine concentration, incubation time, and cell density and was established for routine use with HEK293 cells. The assay was found to be suitable for the testing of novel active site-directed S1P lyase inhibitors, providing important information on their relative potency in intact cells.
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http://dx.doi.org/10.1016/j.ab.2012.11.026DOI Listing
March 2013

A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis.

J Exp Med 2012 Nov 12;209(12):2165-81. Epub 2012 Nov 12.

Medizinische Klinik und Poliklinik I, Klinikum der Universität, Ludwig-Maximilian-Universität München, 81337 Munich, Germany.

Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.
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http://dx.doi.org/10.1084/jem.20121090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3501353PMC
November 2012

BZM055, an iodinated radiotracer candidate for PET and SPECT imaging of myelin and FTY720 brain distribution.

ChemMedChem 2011 Apr 30;6(4):667-77. Epub 2011 Jan 30.

Novartis Institutes for BioMedical Research, Basel, Global Discovery Chemistry, Neurosciences and Molecular Imaging, Postfach, 4002 Basel, Switzerland.

FTY720 (fingolimod, Gilenya®) is a sphingosine 1-phosphate (S1P) receptor modulator that shows significant therapeutic efficacy after oral administration to patients of multiple sclerosis. Because FTY720 does not contain any atom whose PET or SPECT radioisotope would have a half-life compatible with its pharmacokinetic properties, it cannot be used directly for imaging. Instead, we propose BZM055 as a surrogate tracer to study its pharmacokinetics and organ distribution in patients and, given that FTY720 accumulates in myelin sheaths, for myelin imaging. BZM055 (2 a, 2-iodo-FTY720) can be easily radiolabeled with ¹²³I (for SPECT) or ¹²⁴I (for PET). Not only does it closely mimic the pharmacokinetics and organ distribution of FTY720, but also its affinity, selectivity for S1P receptors, phosphorylation kinetics, and overall physicochemical properties. [¹²³I]BZM055 is currently under development for clinical imaging.
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http://dx.doi.org/10.1002/cmdc.201000477DOI Listing
April 2011

Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.

Nat Rev Drug Discov 2010 Nov 29;9(11):883-97. Epub 2010 Oct 29.

Department of Autoimmunity, Transplantation and Inflammation, Novartis Institutes for BioMedical Research, Novartis Campus Forum 1, WSJ-386, CH-4056 Basel, Switzerland.

The discovery of fingolimod (FTY720/Gilenya; Novartis), an orally active immunomodulatory drug, has opened up new approaches to the treatment of multiple sclerosis, the most common inflammatory disorder of the central nervous system. Elucidation of the effects of fingolimod--mediated by the modulation of sphingosine 1-phosphate (S1P) receptors--has indicated that its therapeutic activity could be due to regulation of the migration of selected lymphocyte subsets into the central nervous system and direct effects on neural cells, particularly astrocytes. An improved understanding of the biology of S1P receptors has also been gained. This article describes the discovery and development of fingolimod, which was approved by the US Food and Drug Administration in September 2010 as a first-line treatment for relapsing forms of multiple sclerosis, thereby becoming the first oral disease-modifying therapy to be approved for multiple sclerosis in the United States.
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http://dx.doi.org/10.1038/nrd3248DOI Listing
November 2010

Ovalbumin-induced plasma interleukin-4 levels are reduced in ceramide kinase-deficient DO11.10 RAG1-/- mice.

Lipids Health Dis 2010 Jan 6;9. Epub 2010 Jan 6.

Novartis Institutes for BioMedical Research, Brunnerstrasse 59, A-1235 Vienna, Austria.

Ceramide kinase (CERK) produces the bioactive lipid ceramide-1-phosphate (C1P) and is a key regulator of ceramide and dihydroceramide levels. It is likely that CERK and C1P play a role in inflammatory processes but the cells involved and the mechanisms used remain to be clarified. In particular, the impact of CERK on T-cell biology has not been studied so far. Here, we used Cerk-/- mice backcrossed with DO11.10/RAG1-/- mice to probe the effect of CERK ablation on T-cell activation. Levels of interleukin (IL)-2, IL-4, IL-5, IL-13, of tumor necrosis factor (TNF)-alpha, and of interferon (INF)-gamma were recorded following ovalbumin challenge in vivo and using ovalbumin-treated splenocytes ex- vivo. Absence of CERK led to a significant decrease in the production of IL-4, thus suggesting that CERK may polarize T cells towards the TH2 cell subtype. However, the importance of CERK to TH2 cell biology will have to be investigated further because in a model of asthma, which is TH2-cell driven, Cerk-/- mice responded like wild-type animals.
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http://dx.doi.org/10.1186/1476-511X-9-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2817648PMC
January 2010

4,5,6-Trisubstituted piperidinones as conformationally restricted ceramide analogues: synthesis and evaluation as inhibitors of sphingosine and ceramide kinases and as NKT cell-stimulatory antigens.

Chem Biodivers 2009 Oct;6(10):1688-715

Laboratorium für Organische Chemie, Departement Chemie und Angewandte Biowissenschaften, ETH Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich.

The conformationally based piperidinone sphingosine analogues 7, 8, 15, and 16 were synthesized from allylic alcohol 34 via lactams 31 and 32. The L-arabino diol 7 and the L-ribo diol 8 were transformed into the amino alcohols 17-24. The L-gluco ceramide analogues 43, 46a, and 47, and the L-altro ceramide analogues 51a and 52 were synthesized from either 31 or 32. The L-ribo diols 8 and 16, and the amino alcohols 19 and 20 inhibit sphingosine kinase 1 (SPHK1), while the L-arabino analogues 7, 15, 17, and 18 are inactive. The L-arabino and the L-ribo dimethylamines 21-24, the L-gluco ceramide analogues 43, 46a, and 47, and the L-altro ceramide analogues 51a and 52 did not block SPHK1. Neither the L-arabino diol 7 nor the L-ribo diol 8 inhibited SPHK2 or ceramide kinase. The L-arabino diols 7 and 15 stimulate invariant natural killer T (iNKT) cells when presented by living antigen-presenting cells (APC) and also by plate-bound human CD1d, whereas the L-ribo diols 8 and 16, the L-arabino amino alcohols 17-18, and the dimethylamines 21-22 did not activate iNKT cells. The L-gluco ceramide analogues 43, 46a, and 47 had strongly stimulatory effects on iNKT cells when presented by living APC and also by plate-bound human CD1d, whereas the L-altro ceramide analogue 52 activated only weakly. All activatory compounds induced preferentially the release of pro-inflammatory cytokines, indicating the formation of a stable CD1d--lipid--T-cell receptor complex.
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http://dx.doi.org/10.1002/cbdv.200900045DOI Listing
October 2009

Synthesis and evaluation of sphingolipid analogues: modification of the hydroxy group at C(1) of 7-oxasphingosine, and of the hydroxy group at C(1) and the amide group of 7-oxaceramides.

Chem Biodivers 2009 May;6(5):705-24

Laboratorium für Organische Chemie, Departement Chemie und Angewandte Biowissenschaften, ETH-Zürich, Wolfgang-Pauli-Strasse 10, Zürich.

The analogues 7-9 of 7-oxaceramide and 7-oxasphingosine were synthesized from the known azidosphingosine 21. The 1,4-disubstituted 1,2,3-triazole analogues 10-16 of ceramides were synthesized by the click reaction of the known azide 24. None of the analogues 7-15 was active as inhibitor of SPHK type 1 and of acid sphingomyelinase, whereas 16 is a weak inhibitor of SPHK1. Triazoles 10, 11, and 15 did not inhibit ceramide phosphorylation by CerK, and none of 7, 8, and 10-15 activated invariant natural killer T (iNKT) cell clones when presented by human CD1d-transfected antigen-presenting cells (APC) or by plate-bound human CD1d [55]. Triazoles 14 and 15 prevent binding of alpha-galactosylceramide (alpha-GalCer) to plate-bound human CD1d and subsequent T-cell response to alpha-GalCer. Only 15 reduced activation by alpha-GalCer significantly and independently of the cytokine measured.
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http://dx.doi.org/10.1002/cbdv.200900013DOI Listing
May 2009

Synthesis of 7-aza- and 7-thiasphingosines, and evaluation of their interaction with sphingosine kinases and with T-cells.

Chem Biodivers 2009 May;6(5):725-38

Laboratorium für Organische Chemie, Departement Chemie und Angewandte Biowissenschaften, ETH-Zürich, Wolfgang-Pauli-Strasse 10, CH-8093 Zürich.

The synthesis of 7-oxasphingosine (3) and 7-oxaceramide (4) was improved by starting from the 4-methoxybenzyl-protected d-galactal 9. The sphingosine analogues 5-7 and 24 were synthesized via the azido alcohol 13. The 7-thiasphingosine 5 is a poorer substrate for both isoforms of sphingosine kinase (SPHK) than sphingosine, but showed a slight preference for SPHK2. The sulfone 6 and the 7-aza compounds 7 and 24 were not phosphorylated by either SPHK1 or SPHK2, and none of 5-7 and 24 activated invariant natural killer T (iNKT) cell clones when presented by human CD1d-transfected antigen-presenting cells (APC) or by plate-bound human CD1d. Only 7 and 24 associated with plate-bound recombinant CD1d prevented stimulation of iNKT cells by alpha-galactosylceramide (alpha-GalCer).
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http://dx.doi.org/10.1002/cbdv.200900039DOI Listing
May 2009

Efficient elimination of nonstoichiometric enzyme inhibitors from HTS hit lists.

J Biomol Screen 2009 Jul 21;14(6):679-89. Epub 2009 May 21.

Lead Finding Platform, Protein Structure Unit, Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry, Basel, Switzerland.

High-throughput screening often identifies not only specific, stoichiometrically binding inhibitors but also undesired compounds that unspecifically interfere with the targeted activity by nonstoichiometrically binding, unfolding, and/or inactivating proteins. In this study, the effect of such unwanted inhibitors on several different enzyme targets was assessed based on screening results for over a million compounds. In particular, the shift in potency on variation of enzyme concentration was used as a means to identify nonstoichiometric inhibitors among the screening hits. These potency shifts depended on both compound structure and target enzyme. The approach was confirmed by statistical analysis of thousands of dose-response curves, which showed that the potency of competitive and therefore clearly stoichiometric inhibitors was not affected by increasing enzyme concentration. Light-scattering measurements of thermal protein unfolding further verified that compounds that stabilize protein structure by stoichiometric binding show the same potency irrespective of enzyme concentration. In summary, measuring inhibitor IC(50) values at different enzyme concentrations is a simple, cost-effective, and reliable method to identify and eliminate compounds that inhibit a specific target enzyme via nonstoichiometric mechanisms.
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http://dx.doi.org/10.1177/1087057109336586DOI Listing
July 2009

Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors.

Nat Chem Biol 2009 Jun;5(6):428-34

Developmental & Molecular Pathways and 2Global Discovery Chemistry, Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.

Targeting sphingosine-1-phosphate receptors with the oral immunomodulator drug FTY720 (fingolimod) has demonstrated substantial efficacy in the treatment of multiple sclerosis. The drug is phosphorylated in vivo, and most of the clinical effects of FTY720-phosphate (FTY720P) are thought to be mediated via S1P1 receptors on lymphocytes and endothelial cells, leading to sequestration of lymphocytes in secondary lymphoid organs. FTY720P was described to act as a "functional antagonist" by promoting efficient internalization of S1P1 receptors. We demonstrate here that S1P1 receptors activated by FTY720P retain signaling activity for hours in spite of a quantitative internalization. Structural analogs of FTY720P with shorter alkyl side chains retained potency and efficacy in a functional assay but failed to promote long-lasting receptor internalization and signaling. We show that persistent signaling translates into an increased chemokinetic migration of primary human umbilical vein endothelial cells, which suggests persistent agonism as a crucial parameter in the mechanism of action of FTY720.
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http://dx.doi.org/10.1038/nchembio.173DOI Listing
June 2009

Sphingosine kinase 1 is essential for proteinase-activated receptor-1 signalling in epithelial and endothelial cells.

Int J Biochem Cell Biol 2009 Jul 8;41(7):1547-55. Epub 2009 Jan 8.

Novartis Institutes for BioMedical Research, Brunnerstrasse 59, Vienna, Austria.

There is accumulating evidence that activation of sphingosine kinase 1 (SPHK1) is an important element in intracellular signalling cascades initiated by stimulation of multiple receptors, including certain growth factor, cytokine, and also G-protein coupled receptors. We here report that stimulation of the lung epithelial cell line A549 by thrombin leads to transient increase of SPHK1 activity and elevation of intracellular sphingosine-1-phosphate (S1P); abrogation of this stimulation by SPHK1-specific siRNA, pharmacological inhibition, or expression of a dominant-negative SPHK1 mutant blocks the response to thrombin, as measured by secretion of MCP-1, IL-6, IL-8, and PGE(2). Using selective stimulation of proteinase-activated receptors (PARs) a specific involvement of SPHK1 in the PAR-1 induced responses in A549 cell, including activation of NFkappaB, was evident, while PAR-2 and PAR-4 responses were independent of SPHK1. Moreover, PAR-1 or thrombin-induced cytokine production and adhesion factor expression of human umbilical vein endothelial cells was also seen to depend on SPHK1. Using dermal microvascular endothelial cells from SPHK1-deficient mice, we showed that absence of the enzyme abrogates MCP-1 production induced in these cells upon treatment with thrombin or PAR-1 activating peptide. We propose SPHK1 inhibition as a novel way to block PAR-1 mediated signalling, which could be useful in treatment of a number of diseases, in particular in atherosclerosis.
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http://dx.doi.org/10.1016/j.biocel.2009.01.001DOI Listing
July 2009

Sphingolipid metabolizing enzymes as novel therapeutic targets.

Subcell Biochem 2008 ;49:487-522

Novartis Institutes for BioMedical Research, Brunnerstrasse 59, A-1235 Vienna, Austria.

Pharmacological interference with sphingolipid metabolizing enzymes promises to provide novel ways to modulate cellular pathways relevant in multiple diseases. In this review, we focus on two sphingolipid signaling molecules, sphingosine-1-phosphate (S1P) and ceramide, as they are involved in cell fate decisions (survival vs. apoptosis) and in a wide range of pathophysiological processes. For S1P, we will discuss sphingosine kinases and S1P lyase as the enzymes which are crucial for its production and degradation, respectively, emphasizing the potential therapeutic usefulness of inhibitors of these enzymes. For ceramide, we will concentrate on acid sphingomyelinase, and critically review the substantial literature which implicates this enzyme as a worthwhile target for pharmacological inhibitors. It will become clear that the task to validate these enzymes as drug targets is not finished and many questions regarding the therapeutic usefulness of their inhibitors remain unanswered. Still this approach holds promise for a number of totally new therapies, and, on the way, detailed insight into sphingolipid signaling pathways can be gained.
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http://dx.doi.org/10.1007/978-1-4020-8831-5_19DOI Listing
December 2008

Targeting ceramide metabolism with a potent and specific ceramide kinase inhibitor.

Mol Pharmacol 2008 Oct 8;74(4):925-32. Epub 2008 Jul 8.

Novartis Institutes for BioMedical Research, Vienna, Austria.

Ceramide kinase (CerK) produces the bioactive lipid ceramide-1-phosphate (C1P) and appears as a key enzyme for controlling ceramide levels. In this study, we discovered and characterized adamantane-1-carboxylic acid (2-benzoylamino-benzothiazol-6-yl)amide (NVP-231), a potent, specific, and reversible CerK inhibitor that competitively inhibits binding of ceramide to CerK. NVP-231 is active in the low nanomolar range on purified as well as cellular CerK and abrogates phosphorylation of ceramide, resulting in decreased endogenous C1P levels. When combined with another ceramide metabolizing inhibitor, such as tamoxifen, NVP-231 synergistically increased ceramide levels and reduced cell growth. Therefore, NVP-231 represents a novel and promising compound for controlling ceramide metabolism that may provide insight into CerK physiological function.
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http://dx.doi.org/10.1124/mol.108.048652DOI Listing
October 2008

Binding of pimecrolimus and tacrolimus to skin and plasma proteins: implications for systemic exposure after topical application.

Drug Metab Dispos 2008 Sep 4;36(9):1812-8. Epub 2008 Jun 4.

Novartis Pharma AG, Basel, Switzerland.

Pimecrolimus and tacrolimus are calcineurin inhibitors used for the topical treatment of atopic dermatitis. Although structurally similar, they display specific differences including higher lipophilicity and lower skin permeation of pimecrolimus. The aim of the present study was to understand the reason for the differences in skin permeation; in addition, plasma protein binding of the two drugs was analyzed side by side as a basis for comparison of systemic exposure to free drug. Permeation of pimecrolimus and tacrolimus through a silicon membrane was found to be similar; therefore, we assumed that differences in skin permeation could be caused by differences in affinity to skin components. To test this hypothesis, we investigated binding of pimecrolimus and tacrolimus to a preparation of soluble human skin proteins. One binding protein of approximately 15 kDa, probably corresponding to macrophilin12, displayed a similar binding capacity for pimecrolimus and tacrolimus. However, less specific, nonsaturating binding to other proteins was approximately 3-fold higher for pimecrolimus. Because of the high local drug concentration after topical administration, the unspecific, high-capacity binding is probably dominating the permeation through skin. In plasma both drugs bound predominantly to lipoproteins, which may affect disposition differently from albumin binding. The unbound fraction of pimecrolimus in human plasma was approximately 9-fold lower compared with that of tacrolimus (0.4 +/- 0.1 versus 3.7 +/- 0.8%). In conclusion, these results provide an explanation for the observed lower systemic exposure to pimecrolimus than to tacrolimus after topical application and suggest that differences in systemic exposure to free drug might be even more pronounced.
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http://dx.doi.org/10.1124/dmd.108.021915DOI Listing
September 2008

Phosphorylation by sphingosine kinase 2 is essential for in vivo potency of FTY720 analogues.

ChemMedChem 2008 Jul;3(7):1027-9

Novartis Institutes for Biomedical Research, Brunner Strasse 59, 1235 Wien, Austria.

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http://dx.doi.org/10.1002/cmdc.200800037DOI Listing
July 2008