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Titel:Endothiapepsin und Proteinkinase A: Komplexstrukturen mit neuartigen Inhibitoren, Durchmustern einer Fragmentbibliothek sowie Inhibitordesign ausgehend von einer Sonde
Autor:Köster, Helene
Weitere Beteiligte: Klebe, Gerhard (Prof.)
Veröffentlicht:2012
URI:https://archiv.ub.uni-marburg.de/diss/z2012/0926
DOI: https://doi.org/10.17192/z2012.0926
URN: urn:nbn:de:hebis:04-z2012-09268
DDC:500 Naturwissenschaften
Titel(trans.):Endothiapepsin and Proteinkinase A: Complex Structures of Novel Inhibitors, Screening of a Fragment Library and Probe- based Inhibitordesign
Publikationsdatum:2012-09-25
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Endothiapepsin, Endothiapepsin, protein crystallography, Aspartylprotease . Proteinkristallographie, Proteinkinase A, Arzneimitteldesign, Aspartic proteinase, Proteinkinase A

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.

Summary:
Structure-based drug design is a widely used, successful method in modern drug discovery. To obtain a potent inhibitor an initial lead compound is stepwise optimized to inhibit the target protein. One prerequisite is the knowledge of the binding mode which is normally provided by an X-ray crystal structure. The availability of an initial complex structure is often the time limiting step. The main focus of this work lies on the crystallographic binding mode determination of different ligands. All together 30 protein-ligand complex structures are presented which were determined in the course of two main projects. The first project deals with pepsin-like aspartic proteinases. These proteins play an important role in several severe diseases such as malaria (plasmepsins), Alzheimer’s disease (β secretase) and hypertension (renin). Endothiapepsin served as a model system to investigate the binding mode of several novel ligands. This way the binding mode of inhibitors based on the Gewald reaction, inhibitors with an azepin scaffold and pyrrolidin based inhibitors could be resolved. During this process some interesting details could be observed. In case of the inhibitors based on the Gewald reaction three distinct binding modes were obtained in which the catalytic water molecule plays three different roles: Once it is displaced, once it mediates an interaction towards the ligand as an H-bond acceptor and once as an H-bond donor. The complex structure containing an azepin derivative shows, besides the ligand interacting with catalytic aspartates, two bound fragments of this ligand in the binding pocket. This way almost the entire volume of the binding pocket is occupied which reveals interesting hints on preferred interactions. In the complex structure with the pyrrolidin derivative a disordered wide-open flap with a partly open flap pocket could be observed. This structure therefore provides insights on the flexibility of this protein family. Furthermore, ITC measurements of this complex assured a double deprotonated catalytic dyade. Additionally, complex structures with the clinically applied HIV-protease inhibitors ritonavir and saquinavir were obtained. With respect to ritonavir also a crystal structure with the secreted aspartic proteinase 2 (SAP2) succeeded. While the binding mode of ritonavir is equivalent with the one found in the HIV-protease, saquinavir shows an unusual addressing of the catalytic dyade. Another focus of this project is the fragment-based drug design. Benzamidine and a hydrazine derivative were used as examples to show that small low-affinity ligands can be successfully crystallized. In a second step an inhouse library containing 364 fragments was tested against Endothiapepsin. This led to 55 assay hits. Out of the 55 assay hits for eleven compounds a crystal structure was obtained. The fragments show diverse binding modes and cover almost the entire volume of the binding pocket. Furthermore, this study suggests a critical view on the “rule of three” which is frequently used in the design of fragment libraries. Especially the threshold on the maximal number of H-bond acceptors seems to be too strict. The second project deals with the optimization of a small unspecific probe molecule into a potent inhibitor. The protein kinase A (PKA) is used as an easy to handle model system. Based on phenol, and following several design cycles an inhibitor with an affinity in the low nanomolar range could be synthesized. To achieve this, the approach of a fragment based drug design was combined with the methods of a de-novo design. This study demonstrates how experimental and computer-based methods effective complement each other. Additionally for the first time a rather closed conformation of the glycine rich loop was observed. This provides a valuable starting point for further studies on the dynamic of this protein In summary, the presented crystal structures provide different starting points for further drug design. In case of the pepsin-like aspartic proteinases many novel possibilities to address the catalytic dyad were revealed. Furthermore, the structure showing an open flap in endothiapepsin as well as the one showing a rather closed glycine rich loop in PKA give a first glimpse on the dynamics of these proteins. New methodical aspects are contributed through the conduction of fragment based screenings as well as a probe-based drug design. Last but not least the diverse structures are supportive, especially in combination with computer based and further biochemical methods, to better understand the nature of protein-ligand interactions.

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