Publikationsserver der Universitätsbibliothek Marburg

Titel: Strukturbasiertes Design, Synthese und Affinitätsbestimmung neuartiger HIV-1-Protease-Inhibitoren
Autor: Klee, Nina
Weitere Beteiligte: Diederich, Wibke (Prof. Dr.)
Veröffentlicht: 2013
URI: https://archiv.ub.uni-marburg.de/diss/z2013/0394
URN: urn:nbn:de:hebis:04-z2013-03943
DOI: https://doi.org/10.17192/z2013.0394
DDC: Naturwissenschaften
Titel(trans.): Structure-Based Design, Synthesis and Affinity Determination of novel HIV-1 Protease Inhibitors
Publikationsdatum: 2013-08-20
Lizenz: https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
HIV-Infektion, Chemische Synthese, Bizyklen, Fragment, Protease, Inhibitor, HIV-Proteaseinhibitor, Proteasen, Prodrug, Enzym, HIV, Prodrug, HIV, Enzyminhi, Fragment, Arzneimitteldesign, Organische Synthese, Synthese

Zusammenfassung:
Mit dem humanen Immunschwäche Virus (HIV), dem Verursacher des erworbenen Immunschwäche Syndroms, welches auch als AIDS (Acquired Immunodeficiency Syndrom) bezeichnet wird, sind heute weltweit 34 Millionen Menschen infiziert. Trotz beachtlicher Erfolge mit den 26 aktuell zugelassenen Wirkstoffen im Kampf gegen das Virus, stellen Resistenzen und Nebenwirkungen weiterhin eine große Herausforderung für die Arzneistoffforschung dar. Ein bedeutendes Zielenzym für Virustatika ist die HIV-Protease, ein virales Enzym, welches für die Vermehrungsfähigkeit von HIV essentiell ist. Zurzeit befinden sich 10 zugelassene HIV-Protease-Inhibitoren auf dem Markt. Um die bestehenden Probleme der Verträglichkeit und der Resistenzbildung zu lösen, sind jedoch weiterhin sowohl die akademische als auch die industrielle Forschung gefragt, ihre Anstrengungen zur Entwicklung neuer, besserer HIV-Protease-Inhibitoren fortzusetzen. Da die HIV-Protease, mit über 600 in der PDB (Protein Data Bank) hinterlegten Strukturen, eins der am besten kristallographisch untersuchten Enzyme mit medi¬zinischer Relevanz darstellt, ist das strukturbasierte Wirkstoffdesign eine vielversprechende Methode zur Entwicklung neuer HIV-Protease Inhibitoren. Im Rahmen der vorliegenden Arbeit sollten in einem iterativen Prozess aus strukturbasiertem Design, Synthese und Affinitätsbestimmung neue Inhibitoren der HIV-Protease entwickelt und geeignete Vertreter für einen Zellassay identifiziert werden. Zur Bewertung der Inhibitoren sollte neben dem Ki die Ligandeneffizienz herangezogen werden, da diese eine gute Vergleichbarkeit zwischen Inhibitoren unterschiedlichen Molekulargewichts ermöglicht. Als Zielwert für eine arzneistoffartige (im Englischen druglike) Ver¬bin¬dung gilt eine Ligandeneffizienz von -0,29 kcal/mol, ein Wert der etwa einem 10 nM Inhibitor mit einem Molekulargewicht von ca. 500 g/mol entspricht. Ausgehend von dem sehr potenten aber relativ großen HIV-Protease-Inhibitor AB111 sollten in dieser Arbeit kleinere Inhibitoren der HIV-Protease mit einer verbesserten Ligandeneffizienz strukturbasiert entworfen, dargestellt und auf ihre Enzymaffinität hin untersucht werden. Der erste Ansatz bestand in der Synthese von dreiarmigen Pyrrolidin-basierten-Inhibitoren, welche mit zwei protonierbaren Stickstoffen die katalytische Diade adressieren. Die Ligandeneffizienz konnte gegenüber der Ausgangsverbindung zwar verbessert werden, allerdings ist der Ki der besten Verbindung mit 0,9 µM nur als moderat zu bewerten. Die SAR dieser Verbindungsklasse erwies sich zudem als nicht additiv und auch mit Dockingexperimenten nicht vorhersagbar. Im zweiten Ansatz wurde mit den Oxalsäureamid-Bizyklen ein effizienteres Grundgerüst ent-wickelt, welches mit einem starren Ringsystem sowohl die katalytische Diade der Protease als auch deren Flap-Region adressiert. Bereits die nur zwei Spezifitätstaschen besetzenden Oxalsäureamid-Bizyklen erwiesen sich mit einer Ligandeneffizienz der bes¬ten Verbindung von -0,27 kcal/mol als ausgesprochen vielversprechend, was vermutlich zu einem großen Teil auf einen günstigen entropischen Beitrag des rigiden bizyklischen Ringsystems zur Bindungsenergie zurückzuführen ist. In einem weiteren Optimierungszyklus wurden drei- und vierarmige bizyklische Inhibitoren synthetisiert, um zusätzliche Spezifitätstaschen der HIV-Protease zu besetzen. Drei dieser Verbindungen erreichen oder übertreffen den Zielwert für die Ligandeneffizienz, bleiben aber dennoch mit dem Molekulargewicht in einem günstigen Bereich von unter 500 g/mol. Mit einem Ki von 7 nM liegt die beste der dargestellten Verbindungen zudem in einem sehr guten Affinitätsbereich für einen potentiellen Arzneistoff. Im Zellassay zeigten die ausgewählten bizyklischen Inhibitoren allerdings keine Aktivität, weshalb hier ein Prodrug-Ansatz verfolgt wurde. Carbamatprodrugs der Pyrrolidin-basierten bizyklischen Inhibitoren sollten ungeladen die Zellmembran passieren können und im Zellinneren durch Esterasen, unter Freisetzung des basischen Pyrrolidins, gespalten werden. Die im Zellassay aktivste Verbindung dieser Arbeit stellt ein Methylcarbamat-Prodrug eines vierarmigen bizyklischen Inhibitors dar, welcher eine Zellaktivität von 42 % bei 10 µM aufweist.

Summary:
Today, 34 million people worldwide are infected with the human immunodeficiency virus (HIV), which is the causative agent of the acquired immunodeficiency syndrome (AIDS). The development of numerous antiretroviral drugs, 26 of which are currently approved for treatment of HIV infection, has led to a substantial progress in the combat against HIV and AIDS. However, the development of resistance and the strong adverse effects of the marketed drugs still pose a challenge to drug research. HIV protease is a viral enzyme essential for the replication of HIV and thus constitutes an important target for antiretroviral drugs. Currently, 10 HIV protease inhibitors are available on the market. To solve the steadily increasing problems of resistance and adverse effects of these approved inhibitors, intense academic as well as industrial research efforts are required to further pursue the search for novel, improved HIV protease inhibitors. Since HIV protease represents with over 600 structures deposited in the PDB (Protein Data Bank) one of the crystallographically best studied enzymes with medical relevance, structure-based drug design is a promising tool to find new HIV protease inhibitors. The aim of this thesis was to develop novel HIV protease inhibitors in an iterative process of structure-based design, synthesis, and affinity determination. The most promising compounds were additionally selected for a cytoprotection assay. In addition to the Ki-value, the ligand efficiency was used to assess the synthesised inhibitors, since this measure allows a good comparability of entities of different molecular weight. For a drug-like compound, the desired value for the ligand efficiency is -0.29 kcal/mol, which corresponds to a 10 nM inhibitor with a molecular weight of around 500 g/mol. Starting from the potent but relatively large HIV protease inhibitor AB111, smaller inhibitors with improved ligand efficiency were to be designed, synthesized, and tested for their enzyme affinity within the present thesis. The first approach was to synthesize pyrrolidine-based inhibitors equipped with three substituents addressing the protease’s specificity pockets. These inhibitors address the catalytic dyad of the protease with two protonable amino functions. Through optimisation, the ligand efficiency could be improved compared to the starting compound of this series; however, the best compound with a Ki of 0.9 µM displayed only a moderate enzyme affinity. Additionally, the SAR of this compound class proved to be non-additive and could also not be predicted in docking experiments. In a second approach, pyrrolidino-oxalamides were developed, which possess a rigid core to address the catalytic dyad as well as the flap region of the enzyme and turned out to be a much more efficient scaffold. Even though the inhibitors of the first series only address two of the four specificity pockets, they show largely improved ligand efficiencies, with the best compound displaying a value of -0.27 kcal/mol. These promising results are probably mainly due to the rigid core of the novel scaffold, which most likely leads to an advantageous entropic contribution to the binding affinity. The following optimization cycle resulted in bicyclic inhibitors decorated with three or four substituents addressing the remaining specificity pockets of HIV protease. Three of the synthesized compounds reached or exceeded the target value of -0.29 kcal/mol for the ligand efficiency, displaying a molecular weight under 500 g/mol and a Ki in the low nM range thus depicting the desired properties for a putative drug. Unfortunately, these potent bicyclic compounds displayed no cellular activity. Therefore a prodrug approach was utilized to improve the apparently low cell permeability. Carbamate prodrugs of the pyrrolidine-based bicyclic compounds were synthesized, hence lacking the positively charged nitrogen, which were therefore supposed to better penetrate cell membranes. The carbamates were then envisaged to be cleaved by intracellular esterases to reestablish the active free amino functionality. The cellularly most active compound of the present thesis represents a methylcarbamate prodrug of a bis-benzhydryl-substituted bicyclic inhibitor with a cell activity of 42 % at 10 µM.

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