Structure-based Development of Secondary Amines as Aspartic Protease Inhibitors
Böttcher, Jark
As novel promising scaffold for HIV protease inhibition pyrrolidine-derived inhibitors have recently been reported. In this thesis the stepwise improvement of this compound class to potent inhibitors of wildtype as well as selected mutant proteases utilizing rational drug discovery methods is reported. Based on the crystal structure of a (rac)-3,4-dimethyleneamino-pyrrolidine in complex with HIV-1 protease symmetric pyrrolidine-diesters possessing the same stereochemistry were synthesized following a chiral-pool approach. The most potent compounds of the series achieve one-digit micromolar inhibition towards wild type as well as two mutant proteases (Ile50Val and Ile84Val). The cocrystal structure of one derivative in complex with the Ile84Val HIV protease revealed that two inhibitor molecules are bound in the large active site cavity comprising an area encompassed by the catalytic dyad and the flaps in the open conformation. This is the first HIV protease cocrystal structure in which the open-flap conformation of the enzyme is stabilized by an inhibitor that concomitantly addresses the catalytic dyad. As an alternative approach towards HIV protease inhibitors, the development of symmetric 3,4-bis N-alkyl sulfonamide-pyrrolidines is described. The initial lead structure possessing benzene sulfonamide groups and benzyl substituents exhibited a Ki of 2.2 µM. The X-ray structure in complex with the HIV protease enabled the rational design of a second series of inhibitors and revealed three promising symmetric substitution patterns for further lead optimization: (A) Elongation of the P1/P1’-benzyl moieties with hydrophobic substituents in para-position, (B) ortho-substitution at the P2/P2’-phenyl ring systems, and (C) para-substitution at the P2/P2’-phenyl moieties. All three strategies were pursued and resulted in inhibitors with improved affinities up to 260 nM. To elucidate the underlying factors accounting for the SAR, the crystal structures of four representatives, at least one of each modification type, in complex with HIV protease were determined. These structures provided deeper insights into the protein–ligand interactions and the underlying principles of the SAR thus enabling to choose the most promising combination of substituents in the next design cycle. The combination of these substituents rendered a final inhibitor showing a significantly improved affinity of Ki = 74 nM and the cocrystal structure in complex with the HIV protease confirmed the successful application of the pursued optimization strategy. Subsequently the influence of the active site mutations Ile50Val and Ile84Val on these inhibitors is investigated by structural and kinetic analysis. Whereas the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the crucial Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases the structural basis of this phenomenon was elucidated. Inhibitors bearing smaller N-alkyl substituents revealed a selectivity profile not being explicable with the initial SAR. By cocrystallization of the most potent derivative of a small series with HIV-1 protease, astonishingly two different crystal forms, P2(1)2(1)2(1) and P6(1)22, were obtained. Structural analysis revealed two completely different binding modes, the interaction of the pyrrolidine nitrogen atom to the catalytic aspartates being the only similarity. Encouraged by the successful utilization of cyclic secondary amines as anchoring group in the development of HIV protease inhibitors, this strategy was expanded into a general approach for lead structure identification for aspartic proteases. An initial library comprising eleven inhibitors based on easily accessible achiral linear oligoamines was developed and screened against six selected aspartic proteases (HIV-1 protease, plasmepsin II, plasmepsin IV, renin, BACE-1, and pepsin). Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was consecutively confirmed by determination of the crystal structure of two derivatives in complex with HIV-1 protease. The binding modes exhibit high similarity to the binding orientation of substrates as well as to that of peptidomimetic inhibitors. Using this information, a generalization of this binding situation to other aspartic proteases appears reasonable, thus providing a first insight into the observed structure-activity relationships.
Philipps-Universität Marburg
Chemistry + allied sciences
urn:nbn:de:hebis:04-z2009-00750
https://doi.org/10.17192/z2009.0075
opus:2287
urn:nbn:de:hebis:04-z2009-00750
English
HIV protease
2008
Strukturbasierte Entwicklung von sekundären Aminen als Aspartylprotease-Inhibitoren
Protein crystallography
Philipps-Universität Marburg
Publikationsserver der Universitätsbibliothek Marburg
Universitätsbibliothek Marburg
HIV-Proteaseinhibitor
monograph
Pyrrolidines
2008-11-21
ths
Prof. Dr.
Klebe
Gerhard
Klebe, Gerhard (Prof. Dr.)
Chemistry + allied sciences
Chemie
Molekulardesign
Pharmazeutische Chemie
2011-08-10
https://doi.org/10.17192/z2009.0075
2009-03-03
Strukturbasiertes Wirkstoffdesign
Structure-based inhibitor design
opus:2287
Structure-based Development of Secondary Amines as Aspartic Protease Inhibitors
Proteinkristallographie
Im Rahmen dieser Arbeit wurden neuartige Leitstrukturen für die Inhibition von Aspartylproteasen, insbesondere der HIV-1-Protease, unter Verwendung strukturbiologischer Methoden entwickelt. Als Startpunkt diente die Kristallstruktur eines Komplexes der HIV-1-Protease mit einem 3,4-Dimethylenamino-pyrrolidin-Derivat. Ausgehend von der Beobachtung starker Proteinverzerrung und einer unbefriedigenden Taschenbesetzung in dieser Struktur wurden Verbindungen entwickelt, die nur die essentiellen Pharmacophor-Erfordernisse für einen HIV-Protease-Inhibitor in sich vereinen. Die symmetrischen Pyrrolidindiester-Derivate, ausgestattet mit einer zyklischen sekundären Aminofunktion zur Adressierung der katalytischen Aspartate, zwei hydrophoben Resten für die Besetzung der Erkennungstaschen und zwei Akzeptorfunktionen zur Adressierung der flexiblen Flapregion, zeigten einstellig mikromolare Inhibitionswerte. Die Kristallstrukturanalyse eines Derivates im Komplex mit der Protease offenbarte eine einzigartige Bindungssituation, in der zwei Inhibitormoleküle an ein Protease-Dimer in der geöffneten Konformation binden. Parallel zu diesem Ansatz wurden Verbindungen auf Basis eines 3S,4S-Diamino-pyrrolidin-Gerüstes entwickelt, die über vier Reste zur Adressierung der Subtaschen verfügen. Auch hier zeigten die ersten Verbindungen einstellig mikromolare Inhibition. Aus dem mittels Röntgenkristallographie aufgeklärten Bindungsmodus wurden drei Strategien zur Affinitätssteigerung postuliert und umgesetzt. Alle drei Strategien führten zu Inhibitoren mit einem deutlichen Affinitätsgewinn. Mindestens ein Vertreter jeder Strategie wurde anschließend im Komplex mit der HIV-Protease kristallisiert und analysiert. Aus den beobachteten Bindungsmodi wurden die beiden vielversprechendsten Strategien zur Kombination ausgewählt und in dem finalen Inhibitor vereint. Dieser wies mit 74 nM eine weitere deutliche Affinitätssteigerung auf, und die Analyse der Kokristallstruktur bestätigte den methodischen Ansatz. Mit den deutlich affineren Substanzen wurden kinetische Untersuchungen an zwei ausgewählten, klinisch relevanten Punktmutanten der HIV-Protease durchgeführt. Während ein Affinitätsverlust im Falle der Ile50Val-Mutation zu beobachten ist, zeigen die Verbindungen eine erhebliche Affinitätssteigerung im Falle der Ile84Val-Mutation. Zwei Verbindungen, bei denen dieses Phänomen besonders zu Tage tritt, wurden anschließend im Komplex mit den Protease-Varianten strukturell untersucht. Aus der Analyse ergab sich, dass im Falle der Ile84Val Mutation eine Kombination aus mehreren Faktoren zur beobachteten Affinitätssteigerung beiträgt. Besonders die Beobachtung veränderter physikochemischer Eigenschaften in der Kontaktregion zur Aminosäure 84 ist hierbei von erheblicher Relevanz für die Entwicklung zukünftiger Generationen von HIV-Protease-Inhibitoren, mit einer wahrscheinlich deutlich geringeren Empfindlichkeit gegen diese Mutation. Aus den kinetischen Studien ergab sich zudem, dass Verbindungen mit kleineren Alkylresten ein deutlich verändertes Aktivitätsprofil gegenüber den Mutanten aufweisen. Bei dem Versuch, diese Beobachtung strukturell zu untersuchen, wurden Kokristalle eines in der Zusammensetzung identischen Protein-Ligand Komplexes in zwei verschieden Raumgruppen erhalten. In der orthorhombischen Kristallform ist ein Bindungsmodus vergleichbar zu den vorher untersuchten Verbindungen zu beobachten, wohingegen in der hexagonalen Kristallform ein Bindungsmodus zu beobachten ist, der sich fundamental von diesem unterscheidet. Eine uneinheitliche Struktur-Aktivitäts-Beziehung in Bezug auf die Protease-Varianten legt ein paralleles Vorliegen dieser beiden Bindungsmodi nahe. Anhand der in den Projekten gesammelten Erfahrungen wurde ein genereller Ansatz für die Leitstrukturfindung für Aspartylprotease-Inhibitoren auf der Basis eines Oligoamin-Grundgerüstes entwickelt und umgesetzt. Eine 11 Verbindungen umfassende Bibliothek wurde gegen sechs ausgewählte Proteasen (HIV-1 Protease, Plasmepsin II, Plasmepsin IV, Renin, BACE-1 und Pepsin) getestet und es konnten eine Vielzahl von mikromolare Inhibitoren identifiziert werden. Mit der Hilfe der Kokristallstrukturen zweier Derivate im Komplex mit der HIV-Protease konnte eine generelle Bindungssituation für die untersuchten Aspartylproteasen postuliert und mit Affinitätsdaten bestärkt werden. In dieser Arbeit wurden vielfältige Ansätze für die Entwicklung sekundärer Amine als Aspartylprotease-Inhibitoren erarbeitet. Besonders hervorzuheben ist die Entwicklung von Verbindungen, die an die geöffnete Konformation der HIV-Protease binden und somit die Entwicklung einer neuen Klasse von Inhibitoren ermöglichen. Zudem ist es gelungen, eine neue, potente Klasse von Inhibitoren zu etablieren, die im Vergleich zu den literaturbekannten Inhibitoren über einzigartige Eigenschaften gegenüber der Ile84Val Punktmutation verfügt.
https://archiv.ub.uni-marburg.de/diss/z2009/0075/cover.png
As novel promising scaffold for HIV protease inhibition pyrrolidine-derived inhibitors have recently been reported. In this thesis the stepwise improvement of this compound class to potent inhibitors of wildtype as well as selected mutant proteases utilizing rational drug discovery methods is reported. Based on the crystal structure of a (rac)-3,4-dimethyleneamino-pyrrolidine in complex with HIV-1 protease symmetric pyrrolidine-diesters possessing the same stereochemistry were synthesized following a chiral-pool approach. The most potent compounds of the series achieve one-digit micromolar inhibition towards wild type as well as two mutant proteases (Ile50Val and Ile84Val). The cocrystal structure of one derivative in complex with the Ile84Val HIV protease revealed that two inhibitor molecules are bound in the large active site cavity comprising an area encompassed by the catalytic dyad and the flaps in the open conformation. This is the first HIV protease cocrystal structure in which the open-flap conformation of the enzyme is stabilized by an inhibitor that concomitantly addresses the catalytic dyad. As an alternative approach towards HIV protease inhibitors, the development of symmetric 3,4-bis N-alkyl sulfonamide-pyrrolidines is described. The initial lead structure possessing benzene sulfonamide groups and benzyl substituents exhibited a Ki of 2.2 µM. The X-ray structure in complex with the HIV protease enabled the rational design of a second series of inhibitors and revealed three promising symmetric substitution patterns for further lead optimization: (A) Elongation of the P1/P1’-benzyl moieties with hydrophobic substituents in para-position, (B) ortho-substitution at the P2/P2’-phenyl ring systems, and (C) para-substitution at the P2/P2’-phenyl moieties. All three strategies were pursued and resulted in inhibitors with improved affinities up to 260 nM. To elucidate the underlying factors accounting for the SAR, the crystal structures of four representatives, at least one of each modification type, in complex with HIV protease were determined. These structures provided deeper insights into the protein–ligand interactions and the underlying principles of the SAR thus enabling to choose the most promising combination of substituents in the next design cycle. The combination of these substituents rendered a final inhibitor showing a significantly improved affinity of Ki = 74 nM and the cocrystal structure in complex with the HIV protease confirmed the successful application of the pursued optimization strategy. Subsequently the influence of the active site mutations Ile50Val and Ile84Val on these inhibitors is investigated by structural and kinetic analysis. Whereas the Ile50Val mutation leads to a significant decrease in affinity for all compounds in this series, they retain or even show increased affinity towards the crucial Ile84Val mutation. By detailed analysis of the crystal structures of two representatives in complex with wild-type and mutant proteases the structural basis of this phenomenon was elucidated. Inhibitors bearing smaller N-alkyl substituents revealed a selectivity profile not being explicable with the initial SAR. By cocrystallization of the most potent derivative of a small series with HIV-1 protease, astonishingly two different crystal forms, P2(1)2(1)2(1) and P6(1)22, were obtained. Structural analysis revealed two completely different binding modes, the interaction of the pyrrolidine nitrogen atom to the catalytic aspartates being the only similarity. Encouraged by the successful utilization of cyclic secondary amines as anchoring group in the development of HIV protease inhibitors, this strategy was expanded into a general approach for lead structure identification for aspartic proteases. An initial library comprising eleven inhibitors based on easily accessible achiral linear oligoamines was developed and screened against six selected aspartic proteases (HIV-1 protease, plasmepsin II, plasmepsin IV, renin, BACE-1, and pepsin). Several hits could be identified, among them selective as well as rather promiscuous inhibitors. The design concept was consecutively confirmed by determination of the crystal structure of two derivatives in complex with HIV-1 protease. The binding modes exhibit high similarity to the binding orientation of substrates as well as to that of peptidomimetic inhibitors. Using this information, a generalization of this binding situation to other aspartic proteases appears reasonable, thus providing a first insight into the observed structure-activity relationships.
Arzneimitteldesign
doctoralThesis
167
application/pdf
Pyrrolidinderivate
Fachbereich Pharmazie
Böttcher, Jark
Böttcher
Jark
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