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«The development of activity-based probes for serine proteases Sevnur Serim Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum ...»

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Technische Universität München

Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,

Landnutzung und Umwelt

Lehrstuhl für Chemie der Biopolymere

The development of activity-based probes for serine proteases

Sevnur Serim

Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für

Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung

des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.

Vorsitzender: Univ.-Prof. Dr. D. Langosch Prüfer der Dissertation: 1. TUM Junior Fellow Dr. S. Verhelst

2. Univ.-Prof. Dr. A. Kapurniotu Die Dissertation wurde am __23.10.2013__ bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am __17.02.2014__ angenommen.

Acknowledgements First and foremost, I owe my sincere gratitude to my supervisor Dr. Steven Verhelst for his guidance, giving me the opportunity to step into biochemistry, always having his door open for questions, applying his good aura to my mutants and for being the friend who is always right.

I am deeply grateful to Prof. Dr. Dieter Langosch, my committee chair, for the support especially during the submission process and the opportunity to work in the chair.

This work would have not been possible or at least not as fun without my beloved colleagues of the Verhelst Lab, who not only patiently taught biochemistry to a chemist but also created many memories that I’ll remember for a life time. Many thanks to Olli for being his awesome-self, a constant source of fun (and drumming!) in the lab, along with his all-round scientific creativity and not to forget many beers shared together. Thank you Ute, for all the discussions about life and science, inspiring future plans, starting the chain reaction of running, for the non-judgmental listening ear which I know I can (and I will!) always turn to and making the Zusammenfassung of this thesis actually German. Many thanks to Eliane for making paper writing less frustrating, patiently being the molecular biotechnology supervisor of my students and me, and for being the chatterbox we like. I am thankful to dear Annett, Ute and Eliane for careful proofreading of the thesis. I also would like to thank Christian for many delightful scientific and unscientific conversations and all the movie sessions at his place.

Throughout my PhD I was very lucky to supervise many students who made my work easier. I am grateful to Jonas Lohse, Mathias Leidl, Peter Graf, Melanie Honz, Bettina Prieler and especially Susanne Mayer andPhilipp Baer for their contributions.

I owe thanks to Dr. Oliver Frank from Lehrstuhl für Lebensmittelchemie und molekulare Sensorik for NMR measurements.

iv I would like to acknowledge the members of the chair; Markus, Walter, Yang, Oxana, Jan, Christoph, Chris, Martin, Ellen, Martina, Mark and Ayse for the nice working atmosphere and many shared lunches.

Special thanks are extended to my Munich Turkish friend Sezgin for being the best travel buddy and for the “harmonized” runs; as well as to outside of Munich: my “life coach” Balca for being everything I wanted in a friend, my dearest Banu for best online support, Ayça for being my roomie in the most stressful times, and to my “civilian” friends; Shaughn for endless online encouragement and Christian for many shared rides to the most beautiful places and the language sessions.

And my family… Son ve sonsuz teşekkürler en güzel aileye. Sizin bitmeyen desteğiniz olmadan burda, bu tez ve doktora mümkün olamazdı. Benimle beraber enzimler moleküller öğrendiniz, makaleler yazdınız, laba gelip deneyler yaptınız, tez yazdınız benimle. Nerde olursam olayım hepinizi hep çok seviyorum. Son olarak, olur da bir daha birilerine ithaf edilebilecek bir iş yapmazsam endişesiyle, bu tez anneannem ve dedeme adanmıştır.


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4.1 On-resin strategies for synthesis of diphenyl phosphonate ABPs 56

4.2 Quenched phosphonate ABPs for imaging protease activity 59

4.3 FRET-based assay for inhibitor screening of Lon protease 62

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Curriculum Vitae 141 Abstract Serine proteases play crucial roles in physiological and pathological processes ranging from non-specific digestion to highly regulated functions like cell death, immune response and blood clotting. Like all other proteases, they are synthesized in the cell as inactive zymogens, and are activated often by proteolytic processing.

Once active, their activity is tightly regulated mostly by endogenous protease inhibitors. Therefore, the abundance of a protease does not correspond to its activity.

For a better understanding of enzyme function there has been considerable interest in the design and development of activity-based probes (ABPs).

ABPs are small molecules that covalently bind to the enzymes in an activitydependent manner, distinguishing between active proteases and inactive zymogens or inhibitor bound forms. This allows studying the active fraction of a particular enzyme rather than its overall abundance.

Diphenyl esters of α-aminophosphonates (DPPs) are low molecular weight, irreversible serine protease inhibitors and represent useful reactive head groups for ABPs. While synthetic strategies to generate DPPs in solution have been reported, a major challenge remains to achieve an efficient and rapid way of synthesis. We therefore developed a new synthetic route combining solution and solid phase peptide synthesis, which allows rapid diversification of the recognition moiety and convenient synthesis of DPP ABPs. Using this approach, we generated a small library of diphenyl phosphonate probes. We showed the ability to modulate the reactivity and selectivity of these probes’ and demonstrated activity-dependent labeling of endogenous proteases within a tissue proteome.

In many studies fluorescent reporters have been incorporated into ABPs for target visualization. However, these probes also show fluorescence when free in solution, thus creating high background. To overcome this, quenched activity-based probes have been designed that become fluorescent only after covalent modification of a specific protease target. Here we describe the synthesis and evaluation of the first fluorescently quenched ABPs for serine proteases. Our ABPs carry a fluorophore and a quencher pair, a phosphonate warhead and a guanidinophenyl or a valine recognition element. The probes show a high quenching efficiency, a strong activitydependent reactivity and the expected protease specificity. Real-time imaging experiments of atherosclerosis tissue sections and of neutrophil elastase secreted from primary neutrophils are ongoing.

Apart from ABPs, inhibitors are important tools for assessing protease function in normal and disease states. The ATP-dependent Lon protease lacks specific inhibitors to enable a clear understanding of its mechanism. Lon is a homo-oligomeric heat shock protein which selectively degrades abnormal and damaged proteins, as well as short-lived regulatory proteins and is therefore essential for cellular homeostasis. As bacterial Lon has been shown to be involved in pathogenicity, it has become an important target in the development of novel therapeutic agents. To date, even though few inhibitors of Lon are reported, none of them are highly potent or specific.

We established an in vitro assay to monitor its enzymatic activity and screened for new inhibitors. A peptide substrate of Lon with a fluorophore and a quencher pair was synthesized. The intact peptide is only weakly fluorescent as a result of Förster resonance energy transfer (FRET), whereas an increase in fluorescence can be detected after cleavage by Lon, making it possible to measure and quantify the enzymatic activity. This FRET assay was then used to screen a total of 123 compounds, of which four were found out to have an inhibitory effect against Lon.

Among them we identified thiiranes as a new class of inhibitors of E. coli Lon.


Serinproteasen spielen entscheidende Rollen in physiologischen und pathologischen Prozessen, angefangen von unspezifischer Degradierung von Proteinen bis hin zu stark regulierten Prozessen wie Zelltod, Immunantwort und Blutgerinnung. Wie alle anderen Proteasen werden sie in der Zelle als inaktive Zymogene synthetisiert.

Häufig werden sie ihrerseits durch Proteolyse aktiviert und kurz darauf wieder durch endogene Proteaseinhibitoren gehemmt, wodurch eine strenge Regulation gewährleistet wird. Daher lässt sich von der Menge einer Protease nicht auf ihre Aktivität schließen. Um die Funktionen dieser Enzyme besser untersuchen zu können, besteht ein großes Interesse an der Entwicklung von aktivitätsbasierten Sonden (engl.: activity-based probes, ABPs).

ABPs sind kleine Moleküle, die auf eine aktivitätsbasierte Weise kovalent an Enzyme binden. Sie unterscheiden damit zwischen aktiven und inaktiven Proteasen oder inhibitorgebundenen Formen. Dies ermöglicht die gezielte Untersuchung des aktiven Anteils einer Gruppe eines bestimmten Enzyms anstelle der Gesamtheit.

Diphenylester von α-Aminophosphonaten (DPPs) sind niedermolekulare irreversible Inhibitoren für Serinproteasen, die sich gut als reaktive Kopfgruppen für ABPs eignen. Obwohl bereits Strategien zur DPP-Synthese in Lösung beschrieben wurden, bleibt die schnelle und effiziente Synthese eine Herausforderung. Wir haben einen neuen schnelleren Syntheseweg erschlossen, indem wir synthetische Schritte in Lösung und an der Festphase kombiniert haben. So wird eine schnelle Diversifizierung des Erkennungselements und insgesamt eine einfache Synthese von DPP ABPs ermöglicht. Mit diesem Ansatz wurde eine kleine Bibliothek von DPPSonden hergestellt. Wir konnten zeigen, dass sowohl die Reaktivität als auch die Selektivität der DPP ABPs modulierbar sind. Desweiteren demonstrieren wir die aktivitätsabhängige Markierung von endogenen Proteasen im komplexen Gewebelysat.

Fluoreszente Substituenten dienen der Visualisierung von Targets und werden deshalb oft in ABPs integriert. Allerdings fluoreszieren diese Sonden auch dann, wenn sie frei in Lösung vorhanden sind, und erzeugen so unerwünschte Hintergrundsignale. Um dieses Problem zu überwinden, wurden gequenchte ABPs entworfen, die erst nach kovalenter Bindung an eine spezifische Protease fluoreszieren. Hier beschreiben wir die Synthese und Evaluierung der ersten fluoreszenzgequenchten ABPs für Serinproteasen. Unsere ABPs haben ein Fluorophor und Quencher Paar, ein Phosphonat als reaktive Kopfgruppe und Guanidinophenyl oder Valin als Erkennungselement. Diese Sonden zeigen eine hohe Quencheffizienz, eine starke aktivitätsabhängige Reaktivität und die erwartete Proteasespezifität. Echtzeit-Bildgebung Experimente von atherosclerotischen Gewebe sowie von primären Neutrophilen sekretierter neutrophiler Elastase dauern an.

Neben ABPs sind auch Inhibitoren wichtige Werkzeuge zur Erforschung der Funktionen von Proteasen im normalem und Krankheitszustand. Für die ATPabhängige Lon Protease fehlen spezifische Inhibitoren, die ein klares Verständnis ihres Wirkmechanismus ermöglichen könnten. Lon ist ein homooligomeres Hitzeschockprotein, das selektiv beschädigte sowie kurzlebige regulatorische Proteine abbaut. Daher ist sie von wesentlicher Bedeutung für die zelluläre Homöostase. Seitdem gezeigt wurde, dass bakterielle Lon an der Pathogenität beteiligt ist, gilt sie als ein wichtiges Ziel für die Entwicklung neuer Medikamente.

Bis heute gibt es nur wenige Lon-Inhibitoren, von denen keine hochpotent oder spezifisch sind. Wir haben einen in vitro Assay etabliert um die enzymatische Aktivität der Lon zu messen und nach neuen Inhibitoren zu suchen. Dafür wurde ein Peptidsubstrat für Lon mit einem Fluorophor-Quencher-Paar synthetisiert. Das intakte Peptid fluoresziert nur schwach, da die Energie auf den nichtfluoreszierenden Quencher übertragen wird (Förster-Resonanz- Energie-Transfer, FRET). Nach der Peptidspaltung durch Lon wird dieser Effekt aufgehoben, so dass verstärkte Fluoreszenz gemessen werden kann. Dieses Signal kann als enzymatische Aktivität detektiert und quantifiziert werden. Dieser FRET-Assay wurde verwendet um vier Inhibitoren aus einer Bibliothek von insgesamt 123 Verbindungen zu identifizieren.

Dabei werden Thiirane als eine neue Klasse von Inhibitoren der E. coli Lon identifiziert.

1 Introduction

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1.1 Proteases Proteases, also known as proteolytic enzymes, form one of the largest groups of enzymes with approximately 2% of the total number of proteins present in all types of organisms. They catalyze the irreversible hydrolysis of peptide bonds resulting in the breakdown of proteins into smaller peptides. Proteases are classified according to their mechanism of action:2 Serine, threonine and cysteine proteases take part in covalent catalysis, in which the nucleophile that attacks the peptide bond carbonyl is the hydroxyl of the active site serine/threonine or the thiol of the active site cysteine.

Usually histidine functions as a base. Aspartic, glutamic and metalloproteases, however, function via noncovalent catalysis, where an activated water molecule acts as a nucleophile. Aspartic acid/Glutamic acid residues or metal ions (usually zinc, but also cobalt, manganese, nickel, copper, and iron) serve as acids and bases. The debatable class of asparagine lyases cleave themselves with asparagine as the nucleophile, and this self-cleavage does not involve hydrolysis.3

1.1.1 Serine proteases

Serine proteases are found in all kingdoms of life and constitute the largest group of proteases. They are involved in tightly regulated cascades and signaling events, such as blood coagulation, fibrinolysis, apoptosis, and immune response. Dysregulation of proteolytic activity can lead to pathological conditions like inflammatory diseases, cancer, neurodegenerative and cardiovascular disorders.4 Therefore, the study of both inhibition and activity detection of this group of proteases is of great importance for target identification and drug discovery.

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