A Chemical Biology Investigation of Neutrophil Extracellular Traps
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A Chemical Biology Investigation of Neutrophil Extracellular Traps Zur Erlangung des akademischen Grades eines Doktors de Naturwissenschaften (Dr. ret. nat.) von der Fakultät Chemie der Technischen Universität Dortmund angenommene Dissertation von Masters in Chemistry Nancy E. Martinez aus Amarillo, Texas. United States of America Dekan: Prof. Dr. Roland Winter 1. Gutachter: Prof. Dr. Dr. Herbert Waldmann 2. Gutachter: Prof. Dr. Carsten Watzl 3. Wissenschaftliche Mitarbeiter: Dr. Matthias Müller i This work was carried out under the supervision of Prof. Dr. Dr. Herbert Waldmann at the Faculty of Chemistry of the Technical University of Dortmund and at the Max Planck Institute of Molecular Physiology in Dortmund with close collaboration with Prof. Arturo Zychlinsky at the Max Planck Institute for Infection Biology in Berlin from August 2008 to August 2013. ii iii Dedicated to my parents Daniel and Elvira to my guardian angel Maty, R.I.P. to my family and friends For keeping me grounded while I accomplish my dreams iv v “Everything on Earth has a purpose, every disease an herb to cure it, and every person a mission….” Morning Dove Salish vi vii Abstract viii Neutrophils are short-lived leukocytes that migrate to sites of infection where they phagocytose, degranulate, and form Neutrophil Extracellular Traps (NETs). During NET formation, the nuclear lobules of neutrophils disappear, the chromatin expands and, decorated with neutrophilic granule proteins are expelled. NET formation requires the generation of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, which is activated by the protein kinase C (PKC) activator phorbol-12-myristat-13-acetate (PMA). Bacteria, fungi, viruses, and microbial components and other immune cells are the physiological stimuli of NETs. NETs can be pathogenic, for example in sepsis, cancer, autoimmune and cardiovascular diseases. Therefore, the identification of NET formation inhibitors is of high interest. The focus of the project is the application of chemical biology techniques to study NET formation and identify small molecule inhibitors. A semi-automated high content phenotypic screen was developed using human neutrophils that can identify inhibitors of NET formation. For this purpose, established small molecule compound libraries were screened. The identified small molecule hit compounds were grouped into four categories based on their influence on nuclear morphology and cell viability. These groups are: group 1: viable cells with small nuclei; group 2: viable cells with large nuclei; group 3: dead cells with small nuclei; and group 4: dead cells and NET formation. Since this is the first assay of NETosis to be reported, it was patented. After the screening of commercial compound libraries, GW5074, a c-Raf kinase inhibitor that stopped NET formation with group 1 characteristics was identified. To investigate if the Raf-MEK- ERK pathway is involved in NET formation, specific kinase inhibitors, GW5074 (c-Raf), U0126 (MEK), Staurosporine (PKC) kinase inhibitors and the ERK inhibitory peptide (ERK) were tested. They were all identified as group 1 phenotype inhibitors. Ras, an upstream protein from Raf, was excluded as a key protein involved in the NET formation mechanism after testing indirect Ras inhibitors. Additionally, the involvement of the ERK pathway upstream of NADPH oxidase was proven. It was shown that anti-apoptotic proteins are expressed to allow NETosis to occur and are regulated by MAPK signaling pathway. The neutrophils phagocytic and degranulation functions were shown to be uneffected in the presence of inhibitors. This work was published in Nature Chemical Biology (Hakkim et al., 2011). In addition to the ERK signaling pathway, the involvement of the eicosanoid pathway in NET formation was established. Arachidonic acid, diaglycerol and platelet activation factor, known activators of the eicosanoid pathway, were identified as triggers of NET formation. Furthermore, it was demonstrated that lipoxygenase (LOX) is required for NETosis by using specific LOX inhibitors that blocks NET formation. The COX signaling pathway was excluded due to the lack of inhibitory activity of the COX specific compounds. Traditionally in innate immunology, lipid signalling is known ix to be involved in cell-to-cell communication. For NETosis, the involvment of this role it was confirmed by the identification of lipid bodies present on the NETs. It is hypothesized that the lipid bodies are composed of a signaling lipid. This work needs to be further established and is beyond the scope of this thesis. Our results elucidated a new function in leukocytes, leading to possibilities to develop a novel field in neutrophil biology. After screening the MPI Dortmund in-house-compound library, tetrahydroisoquinolines (THIQs) were identified as a novel class of NET formation inhibitors. THIQs inhibit NETosis at low micromolar concentration independent of the NET formation stimulus and at different stages of NET formation. Cells treated with THIQ 1 displayed decondensed nuclei and the phenotype was assigned to group 2, making this an inhibitor with a unique profile. As before, the influence of THIQs on the oxidative burst was studied. THIQs do not affect ROS formation suggesting that they act downstream of ROS formation. Next, the THIQs were biochemically tested on proteins that are involved in the mechanism of NET formation. Interestingly, none of these proteins were shown to be direct targets for the THIQs. Based on the structure activity relationship (SAR) of 100 THIQs, tool compounds suited for target identification were designed and synthesized. With these compounds, affinity-based proteomic experiments were performed on human neutrophil lysates. Unfortunately, no proteins were identified that were selectively enriched with the active probe in comparison to the control. The cellular target of the THIQs might not be a cytosolic soluble protein and is yet to be identified. A hallmark of many immune disorders is the overproduction and lack of degradation of NETs. NET formation inhibitors are considered promising candidates for the treatment of one autoimmune disease, systematic lupus erythematosus (SLE). Therefore, SLE patient-derived neutrophils were used to evaluate the ability of THIQs to inhibit NET formation in a physiologically highly relevant setting. THIQs inhibited NET formation efficiently in non-activated and PMA- activated SLE patient neutrophils. These results clearly indicate that THIQs have great potential as tool compounds and as a starting point for the development of novel small molecule therapeutics. The study of the neutrophil NET formation enigma is a perfect example wherein a chemical biology approach can be applied successfully. A robust assay protocol was established allowing NET formation to be studied quickly and could be scaled up to a high throughput setup. Two novel signalling mechanisms were identified and added to the overall mechanism of NETosis. A novel small molecule NET inhibitor compound class was identified and added to the general toolbox developed by us and others to study NETs. x xi Zusammenfassung xii Neutrophile sind kurzlebige Leukozyten, die zu Infektionsorten wandern, wo sie phagoyztieren, degranulieren und sogenannte Neutrophil Extracellular Traps (NETs, deutsch: Neutrophile Extrazelluläre Fallen) bilden. Während der NET-Formation lösen sich die nuklearen Lobuli der Neutrophilen auf, das Chromatin dehnt sich aus und wird zusammen mit neutrophilen Granulaproteinen in den extrazellulären Raum ausgestoßen. Bakterien, mikrobielle Komponenten, Pilze, Viren und andere Immunzellen gehören zu den physiologischen Aktivatoren der NET- Formation. Die Bildung von NETs kann außerdem durch den Proteinkinase C (PKC) Aktivator Phorbol-12-myristat-13-acetat (PMA) induziert werden, der die Nicotinamid-Adenin- Dinukleotidphosphat (NADPH) -Oxidase katalysierte Bildung von reaktiven Sauerstoffspezies (ROS) und so letztlich NET-Formation bewirkt. Die Formation von NETs kann auch in Krankheiten involviert sein, unter anderem in Sepsis, Krebs und Autoimmun- sowie Herz-Kreislaufkrankheiten. Aus diesem Grund ist die Identifizierung von NET-Bildungsinhibitoren von großem Interesse. Der Schwerpunkt dieses Projektes ist es kleine Moleküle, welche die NET-Formation inhibieren zu identifizieren und diese im Sinne eines Chemischen Biologie basierten Ansatzes zu nutzen um die Formation von NETs zu genauer zu untersuchen. Ein semi-automatisierter, multiparametrischer phenotypischer Screen zur Identifizierung von Inhibitoren der NET-Formation in humanen Neutrophilen wurde entwickelt und mehrere Substanzbibliotheken wurden in dem neuartigen Assay getestet. Die aktiven Substanzen wurden dann, basierend auf dem von ihnen induzierten Phänotypen in eine von vier Kategorien eingeteilt. Diese Kategorien waren: 1) Lebende Zellen mit kleinem Nucleus, 2) Lebende Zellen mit großem Nucleus, 3) Tote Zellen mit kleinem Nucleus, und 4)Tote Zellen die NETs gebildet haben. Dieser Assay war der erste seiner Art und wurde daher patentiert. In dem Screening von kommerziellen Substanzbibliotheken wurde GW5074, ein bekannter c-Raf Kinase Inhibitor, als Inhibitor der NET-Formation der Kategorie 1 identifiziert. Daher wurden weitere Inhibitoren des Raf-MEK-ERK Signalwegs auf ihren Effekt auf die NET-Formation hin untersucht. Die getesteten Substanzen GW5074 (c-Raf Inhibitor), U0126 (MEK-Inhibitor), Staurosporin (PKC Inhibitor) und das ERK inhibitory peptide (ERK Inhibitor) induzierten