Dokument

Zusammenfassung:
Das strukturbasiertes Wirkstoffdesign ist eine vielgenutzte, erfolgreiche Methode neue Arzneistoffe zu entwickeln. Mit dem Ziel einen hochaffinen Inhibitor zu erhalten, wird eine Verbindung schrittweise an ein Zielprotein angepasst. Eine Grundvoraussetzung für ein solches Design ist die Kenntnis des Bindungsmodus. In der Regel wird dazu eine Protein-Ligand-Komplexstruktur herangezogen. Die Verfügbarkeit einer initialen Komplexstruktur, welche meist röntgenkristallographisch erhalten wird, ist nicht selten der zeitbestimmende Schritt. Die kristallographische Aufklärung der Bindungsmodi unterschiedlicher Liganden ist der Schwerpunkt dieser Arbeit. Es werden insgesamt 30 Ligand-Protein Komplexstrukturen vorgestellt, die im Rahmen zweier Projekte erhalten wurden. Das erste Projekt behandelt pepsinähnliche Aspartylproteasen als Zielproteine. Pepsinähnliche Aspartylproteasen spielen eine entscheidende Rolle bei einigen schwerwiegenden Erkrankungen, wie z.B. Malaria (Plasmepsine), Alzheimer (β Sekretase) und Bluthochdruck (Renin). Mit Endothiapepsin als Modellprotein wurden verschiedene neuartige Liganden untersucht. Auf diese Weise konnte der Bindemodus von Inhibitoren basierend auf der Gewaldreaktion und solchen mit einem Azepin- sowie Pyrrolidingrundgerüst aufgeklärt werden. Dabei wurden einige überraschende Details beobachtet. So wurden für die auf der Gewaldreaktion basierenden Inhibitoren drei grundverschiedene Bindungsmoden erhalten, in denen das katalytische Wasser in drei verschiedenen Rollen beobachtet wurde: einmal wird es verdrängt, einmal vermittelt es eine Interaktion zum Liganden als Wasserstoffbrücken-Akzeptor und einmal als Donor. Die Komplexstruktur mit einem Azepin-Derivat zeigt neben dem an die beiden Aspartate gebundenen Liganden zwei Bruchstücke desselben in der Bindetasche. Dadurch ist nahezu die gesamte Bindetasche besetzt, was wertvolle Hinweise auf bevorzugte Interaktionspunkte liefern kann. In der Komplexstruktur mit einem Pyrrolidin findet sich eine ungeordnete, weit geöffnete Flap-Region mit einer teilweise geöffneten Flap-Tasche. Diese Struktur gewährt damit einen Einblick in die Flexibilität dieser Proteine. Zudem konnte anhand dieses Komplexes mittels ITC eine zweifach deprotonierte katalytische Diade nachgewiesen werden. Darüber hinaus wurden Komplexstrukturen mit den klinisch relevanten HIV-Protease Inhibitoren Ritonavir und Saquinavir erhalten, wobei mit Ritonavir auch eine Komplexstruktur mit der sekretorischen Aspartylprotease 2 (SAP2) gelang. Während der Bindemodus von Ritonavir dem in der HIV-Protease entspricht, zeigt Saquinavir eine ungewöhnliche Adressierung der katalytischen Diade. Ein weiterer Schwerpunkt dieses Projekts ist das fragmentbasierte Design. Nachdem anhand von Benzamidin sowie eines Hydrazons gezeigt wurde, dass auch kleine Verbindungen mit schwacher Affinität erfolgreich kristallisiert werden können, wurde eine hauseigene 364 Fragmente umfassende Bibliothek gegen Endothiapepsin getestet. Von den 55 Assayhits konnte von elf Fragmenten eine Komplexstruktur erhalten werden. Die Fragmente zeigen dabei vielfältige Bindungsmodi und decken einen Großteil der Bindetasche ab. Zudem regt diese Untersuchung an, die für die Zusammenstellung vieler Fragmentbibliotheken angewandte Dreierregel, insbesondere die Anzahl der erlaubten Wasserstoffbrücken Akzeptoren, kritisch zu hinterfragen. Das zweite Projekt befasst sich mit der exemplarischen Optimierung einer kleinen unspezifischen Sonde zu einem hochaffinen Inhibitor. Dazu wurde die Proteinkinase A als einfach zu handhabendes Modellprotein ausgewählt. In mehreren Zyklen konnten ausgehend von Phenol durch eine Kombination der Methoden des fragmentbasierten sowie des de-novo Designs Inhibitoren mit einer Affinität im niedrig nanomolaren Bereich synthetisiert werden. Diese Studie demonstriert, wie sich experimentelle und computergestützte Methoden wirkungsvoll ergänzen. Darüber hinaus konnte erstmals eine weitgeschlossene Konformation der glycinreichen Schleife beobachtet werden. Zusammenfassend bieten die in dieser Arbeit vorgestellten Strukturen unterschiedliche Ansatzpunkte für eine weitere Wirkstoffentwicklung. So wurde für die pepsinähnlichen Aspartylproteasen eine Vielzahl neuer Möglichkeiten zur Adressierung der katalytischen Diade aufgezeigt. Zudem helfen die hier vorgestellten Strukturen einer geöffneten Flap von Endothiapepsin sowie einer weitgeschlossenen glycin-reichen Schleife der PKA die Dynamik dieser Proteine besser zu verstehen. Methodisch wurden neue Impulse zur Durchführung von fragmentbasierten Screenings sowie die Möglichkeit eines sondenbasierten Designs vorgestellt. Und nicht zuletzt helfen die vielfältigen Strukturen, insbesondere in Kombination mit computergestützten und weiteren biochemischen Methoden, die Natur von Ligand-Protein Wechselwirkungen besser zu verstehen.

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