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Target Identification of Natural Products by Activity Based Protein Profiling and a Whole Proteome Inventory of Background Photocrosslinker Binding

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Target Identification of Natural Products by Activity Based Protein Profiling and a Whole Proteome Inventory of Background Photocrosslinker Binding TECHNISCHE UNIVERSITÄT MÜNCHEN FAKULTÄT CHEMIE LEHRSTUHL FÜR ORGANISCHE CHEMIE II Target identification of natural products by activity based protein profiling and A whole proteome inventory of background photocrosslinker binding Wolfgang Heydenreuter Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat) genehmigten Dissertation. Vorsitzender: Prof. Dr. Lukas Hintermann Prüfer der Dissertation: 1. Prof. Dr. Stephan A. Sieber 2. Prof. Dr. Tobias A.M. Gulder Die Dissertation wurde am 06.07.2016 bei der bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 28.07.2016 angenommen. TECHNISCHE UNIVERSITÄT MÜNCHEN FAKULTÄT CHEMIE LEHRSTUHL FÜR ORGANISCHE CHEMIE II Target identification of natural products by activity based protein profiling and A whole proteome inventory of background photocrosslinker binding Vollständiger Abdruck zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat) Wolfgang Heydenreuter Meiner Familie gewidmet “Success is not final, failure is not fatalμ it is the courage to continue that counts.” ― Winston S. Churchill Danksagung Zu allererst möchte ich mich sehr herzlich bei Prof. Dr. Stephan A. Sieber für die Aufnahme in seinen Arbeitskreis bedanken. Für sein stetiges Vertrauen in meine Arbeit, die Aufgeschlossenheit gegenüber neuer Projekte und besonders für seine große Begeisterung für die Forschung und die mir gegebenen Freiheiten bei der Entwicklung eigener Projekte bin ich sehr dankbar. Bedanken möchte ich mich auch bei den Mitgliedern der Prüfungskommission für ihre Bemühungen bei der Bewertung dieser Arbeit. Die vielen Ergebnisse wären nie ohne die großartige Zusammenarbeit mit Kooperations- partnern sowohl in als auch außerhalb des Sieber-Labs entstanden. Ganz besonders möchte ich Simone Braig und ihren Mitarbeitern vom Lehrstuhl für Pharmazeutische Biologie der LMU für die ausgesprochen produktive Zusammenarbeit im Rahmen des Neocarzilin–Projektes bedanken. Meinen Laborkollegen Philipp Kleiner, Elena Kunhold, Matthias Stahl, Jan Vomacka, Megan Wright und Vadim Korotkov danke ich für die gemeinsame Arbeit an diversen Projekten im Zuge meiner Doktorarbeit. Mein großer Dank gilt ebenso Megan Wright und Matthias Stahl für ihre Bemühungen dieser Arbeit die Mäkel auszutreiben. Für ihre jahrelange Unterstützung im Laboralltag danke ich ganz besonders Mona Wolff und Katja Bäuml, die durch ihren unermüdlichen Einsatz stets eine produktive Arbeitsumgebung aufrechterhalten haben. Ferner möchte ich mich bei den unzähligen Praktikanten (Forschungspraktikum oder Bachelorarbeit) bedanken, die durch ihr stetiges Engagement viele, besonders synthetische Projekte, maßgeblich vorangetrieben haben. Es ist schön zu sehen, dass einige nun fester Bestandteil des Sieber-Labs geworden sind. Bei meinen Laborkollegen möchte ich mich für die großartige Zeit und deren Hilfsbereitschaft bedanken. Besonders erwähnen möchte ich hier Dr. Georg “Eure Dudeheit“ Rudolf für legendäre Kino- und Bouldersessions sowie philosophische Diskussionen über die emotionale Tiefe von Sharknado-Filmen, Dr. Jürgen “Fernet Branca“ Eirich für entspannte Golfsessions an der Isar und das Belächeln rheinischer Golfkünste, Dr. Maria “Marie“ Dahmen für die Präsentation dieser Künste und diverse Kletterhallenbesuche, Dr. Roman Kolb für das Vorleben von Sarkasmus und Zynismus in vollendeter Form, Volker “Rául von Schalke“ Kirsch und Christian “Fetzle“ Fetzer für exzellente Zockersessions und rege Diskussion über die Signifikanz bakterieller Proteasen. So isch lässig ! Des Weiteren danke ich Philipp Kleiner, Annabelle “Anni“ Hoegl, Johannes “Leeeman“ Lehmann, Mathias “Horst Seeler“ Hackl, Elena “Lenchen“ Kunhold, Dr. Franziska “Franjo“ Mandl, Dr. Max Koch, Dr. Megan Wright, Dr. Pavel Kielkowski, Barbara “Babsarella“ Hofbauer, Thomas “Elfmetergott“ Gronauer, Markus Lakemeyer, Matthias Stahl, Weining Zhao, Jan Vomacka, Dr. Nina Bach, Dóra Balogh, Dr. Joanna Krysiak, Dr. Malte Gersch, Dr. Matthew Nodwell, Dr. Johannes Kreuzer, Dr. Thomas Menzel, Dr. Vadim Korotkov und Dr. Tanja Wirth für die tolle Atmosphäre und die Unterstützung über all die Jahre. Der aktuellen Generation von Masterstudenten namentlich Johnny Drechsel, Ines Hübner und Anja Fux wünsche ich auf ihrem künftigen Weg alles Gute. Mein größter Dank gilt meinen Eltern, Gisela und Peter Heydenreuter, sowie meiner Schwester Christine für deren bedingungslose Unterstützung über all die Jahre. Auch möchte ich mich ganz besonders bei Isabel Buchsbaum für ihren liebvollen Rückhalt in den letzten drei Jahren bedanken. Ich kann mich glücklich schätzen, dass es euch gibt. ________________________________________________________________________________ Table of Contents Table of Contents I Introductory Remarks IV Summary V Zusammenfassung VI 1 Introduction 1 1.1 Activity based protein profiling (ABPP) 1 1.2 Electrophilic natural products and covalent inhibitors 5 1.3 Scope of this work 6 2 Target identification of falcarinol in human cancer cells 9 2.1 Polyynes and falcarinol 9 2.2 ALDH2 10 2.3 Results and discussion 12 2.3.1 Synthesis of activity-based probes 12 2.3.2 Total synthesis of falcarinol and stipudiol 13 2.3.3 Bioactivity in eukaryotic cells and bacteria 15 2.3.4 Protein reactivity in human cancer cells 16 2.4 Concluding remarks and outlook 22 3 A whole proteome inventory of background photocrosslinker binding 23 3.1 Photocrosslinkers 23 3.2 Results and discussion 26 3.2.1 Synthesis of minimal photoprobes 26 3.2.2 Protein reactivity in human cancer cells A549 and HeLa 27 3.2.3 Gel-free analysis of photocrosslinker-specific targets via quantitative proteomics 28 3.2.4 Diazirine-specific photome 30 3.2.5 Target analysis of the PKA inhibitor H8 as a proof-of-principle 34 3.3 Conclusion and outlook 38 4 Target identification of neocarzilin A-C in human cancer cells 39 4.1 Cancer as a challenge for mankind 39 4.1.1 Tumor migration 40 4.1.2 Neocarzilin 41 4.2 Results and discussion 41 4.2.1 Synthesis of an activity-based probe 41 4.2.2 Synthesis of neocarzilins A-C 42 4.2.3 Reactivity to proteins in human cancer cells 43 4.2.4 Quantitative proteomics 45 4.2.5 Target validation 47 4.2.5.1 Labeling of VAT-1 knockdown cells 47 4.2.5.2 Labeling of recombinant VAT-1 in E. coli 48 4.2.6 Function of VAT-1 48 4.2.7 Inhibition of migration and chemotaxis by neocarzilin A 49 4.2.8 Effects of neocarzilin A and C on cellular viability and apoptosis 50 I ________________________________________________________________________________ 4.2.9 Effect of neocarzilin A on mitochondria 52 4.2.10 Identification of potential interaction partners of VAT-1 by co- immunoprecipitation 54 4.3 Conclusion and outlook 57 5 Experimental Section 59 5.1 Organic Synthesis 59 5.1.1 General Methods and Materials 59 5.1.2 Synthetic procedures for chapter 2: Target identification of falcarinol in cancer cells. 60 5.1.3 Synthetic procedures for chapter 3: A whole proteome inventory of background photocrosslinker binding 78 5.1.4 Synthetic procedures for Chapter 4: Target identification of neocarzilin A-C in human cancer cells. 88 5.2 Biochemistry 98 5.2.1 General methods and materials 98 5.2.1.1 Cell culture 98 5.2.1.2 MTT- Assay 98 5.2.1.3 Labeling reagents 100 5.2.1.4 In situ labeling 100 5.2.1.5 Analytical gel based analysis 101 5.2.2 Biochemical procedures for Chapter 2: Target identification of falcarinol in cancer cells 101 5.2.2.1 Click reaction and preparative gel-free analysis 101 5.2.2.2 Preparative gel based analysis 102 5.2.2.3 Gel based target identification 104 5.2.2.4 Gel free target identification 105 5.2.2.5 Binding peptide identification 106 5.2.2.6 Binding site identification 106 5.2.2.7 Recombinant expression of target protein 107 5.2.2.8 PCR 107 5.2.2.9 Gateway cloning 108 5.2.2.10 ALDH2 in vitro-labeling 108 5.2.2.11 ALDH2 activity assay 109 5.2.3 Biochemical procedures for Chapter 3: A whole proteome inventory of background photocrosslinker binding 109 5.2.3.1 Analytical in situ AfBPP labelling 109 5.2.3.2 Preparative in situ labelling and quantification via SILAC 110 5.2.3.3 Mass spectrometry 111 5.2.3.4 Bioinformatics 112 5.2.4 Biochemical procedures for Chapter 4: Target identification of neocarzilin A- C in human cancer cells 114 5.2.4.1 Click reaction and preparative gel-free analysis 114 5.2.4.2 Preparative gel free analysis using label free quantification 114 5.2.4.3 Mass spectrometry 115 5.2.4.4 Recombinant expression of target protein 116 5.2.4.5 PCR 117 5.2.4.6 Gateway cloning 117 5.2.4.7 Recombinant expression and analytical labeling in E.coli 118 5.2.4.8 Co-Immunoprecipitation 119 6 Bibliography 121 7 List of abbreviations, acronyms and symbols 141 II ________________________________________________________________________________ 8 Appendices 144 8.1 Selected 1H and 13C NMR Spectra for Chapter 2 144 8.2 Selected 1H and 13C NMR Spectra for Chapter 4 149 9 Curriculum Vitae 154 III ________________________________________________________________________________ Introductory Remarks The present doctoral dissertation was accomplished between February 2012 and July 2016 under the supervision of Prof. Dr. Stephan A. Sieber at the Chair of Organic Chemistry II of the Technische Universität München. Parts of this thesis are based on the following publications and manuscripts in preparation for publication: Chapter 2: Heydenreuter, W., Kunold, E. & Sieber, S. A. Alkynol natural products target ALDH2 in cancer cells by irreversible binding to the active site. Chem. Commun. (Camb) 51, 15784-15787 (2015). Chapter 3: Kleiner, P., Heydenreuter, W., Stahl, M., Korotkov, V. S., Sieber, S. A. A whole proteome inventory of background photocrosslinker binding. Submitted to Angewandte Chemie. Chapter 4: Manuscript in preparation. All compounds mentioned in the theoretical part of this thesis are indexed consecutively with Arabic numerals in the format X.Y while X being the number of the project chapter and Y being in the order of appearance.
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