Publikationsserver der Universitätsbibliothek Marburg

Titel:Fragment based Drug Discovery; Design and Validation of a Fragment Library; Computer-based Fragment Screening and Fragment-to-Lead Expansion
Autor:Craan, Tobias Friedrich
Weitere Beteiligte: Klebe, Gerhard (Prof. Dr.)
Veröffentlicht:2011
URI:https://archiv.ub.uni-marburg.de/diss/z2011/0429
URN: urn:nbn:de:hebis:04-z2011-04293
DOI: https://doi.org/10.17192/z2011.0429
DDC: Naturwissenschaften
Titel(trans.):Fragment-basierte Wirkstoffentwicklung; Entwicklung und Validierung einer Fragmentbibliotek; Computer-basierte Fragment-Suchen und Fragment zu Leitstruktur Entwicklung
Publikationsdatum:2011-06-28
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Fragmentbibliotek, FBLD, Fragment, Fragment library, Fragmentbasierte Leitstrukturentwicklung

Summary:
In recent years, fragment screening has become a popular approach to identify new lead structures. Fragments are usually defined by the Astex ‘rule of three’ (RO3). Surface Plasmon Resonance (SPR), Nuclear Magnetic Resonance spectroscopy (NMR), biochemical assays and X-ray crystallography are efficient screening techniques to discover prospective fragments as binders. However, these methods need an assembled fragment library. We designed an in-house fragment library, starting from approx. 380,000 commercially available fragments. During library design, we modified the RO3 and we did no strict filtering of physico-chemical properties during fragment enumeration (e.g. twice the number of H-bond acceptors was allowed). The fragments were stepwise reduced to 4,000 compounds. The last step was a visual inspection of the candidates, which lead to a final fragment library of 364 fragments. To validate the quality of the library, we screened it against endothiapepsin. The biochemical screening suggested 55 hits, which were entered into a crystallographic screen. Eleven complex crystal structures were determined, pointing out the remarkably high hit rate of the designed library. HotspotsX is a program which predicts (based on knowledge-based potentials) the probability of a certain atom type at a certain position in the binding pocket of a target enzyme. The eleven crystal structures obtained before were used to validate the program HotspotsX. Due to chemical diversity and the different binding modes of the fragments observed for the library examples we obtained binding through aromatic- , H-bond donor- , acceptor- , doneptor- and hydrophobic interactions. The calculated HotspotsX maps coincide remarkably well with the crystallographically determined fragment positions inside the binding pocket. The program HotspotsX has also been validated with crystal structures of molecular probes like phenol, urea and methylurea. Crystal structures of these molecular probes were determined with different targets. Overall, the experimental hotspot analysis coincided well with the computed contour maps. Thus, the calculated maps by HotspotsX have an excellent predictive power. Based on the binding modes of the molecular probe phenol to the cAMP-dependent protein kinase A (PKA), we started a fragment growing approach. In the latter complex, three phenol molecules are bound. Two are occupying the ATP binding site and one is sitting on top of the glycine-rich loop (G-loop). A virtual screening, using the hinge binding phenol as constraint, suggested a phenol derivative for which a crystal structure could be determined. Starting from this hit, a hotspot analysis was performed. This analysis indicates that growth in the direction of the G-loop, placing an aromatic portion under the G-loop and an acceptor functionality capable to address Lys72 is desired. The first compound of this de novo design had an affinity of 70 µM. In the following first design cycle, we were able to enhance the affinity to 6.5 µM. In the second design cycle an additional amino function was introduced, which did not improve affinity dramatically, but enhanced ligand efficiency to 0.38. In the last cycle, a spacer of one and two methylene groups was introduced and the affinity could be increased to about 110 nM for a diastereomeric mixture of four compounds. The phenol-PKA complex provides a putative allosteric site of PKA. The G-loop in this structure is in a closed state which is stabilized by two H-bonds. This G-loop conformation is probably induced by the phenol molecule sitting on top of the G-loop. Therefore, several molecular dynamics (MD) studies were performed, lacking different phenol molecules, to get insights into the G-loop opening. The MD studies suggest that after removal of the phenol sitting on top of the G-loop some first side chain movements are initiated that can indicate the first steps of the G-loop opening cascade. In a different project, a virtual screening approach was used to find new inhibitors for aldose reductase. A pre-filtered subset of the ZINC database was used as ligand dataset. For the best hit, a series of five compounds was synthesized. Among them one compound displayed an inhibition of 920 nM. The available assays to detect fragment hits are currently not sufficient. The challenges are the low affinity of the fragments and their poor solubility. Therefore, the known thermal shift assay was applied and adapted to detect fragment hits. To validate the method, it was used to characterize variant mutations of EctD. Lastly, a modeling study was used to get ideas about possible binding modes of arachidonic acid derivatives in a K+ ion channel. One predominant binding pose could not be suggested. The study proposes, however, that one arachidonic acid molecule can occupy the inner pore cavity, which is consistent with experimental data.

Zusammenfassung:
In den letzten Jahren wurden vermehrt Fragment-basierte Verfahren verwendet, um neue Leitstrukturen zu identifizieren. Fragmente werden anhand der Astex-Dreier-Regel definiert. SPR, NMR, sowie biochemische Assays und Röntgenstrukturanalyse sind effiziente Verfahren um Fragmente zu entdecken. Diese Methoden müssen auf eine bestehende Fragmentbibliothek angewendet werden. Wir haben unsere eigene Fragmentbibliothek entwickelt. Während des Designs der Fragmentbibliothek haben wir die Astex-Dreier-Regel modifiziert. Es wurde kein strikter physiko-chemischer Filter verwendet. Im letzen Schritt wurden sie visuell inspiziert, was zu einer Bibliothek mit 364 Fragmenten führte. Um die Güte der Bibliothek zu überprüfen, haben wir diese gegen Endothiapepsin getestet. Als Ergebnis erhielten wir 55 aktive Fragmente. Diese wurden kristallographisch untersucht, wobei elf Kristallstrukturen bestimmt werden konnten. Das Programm HotspotsX kann aufgrund von wissensbasierten Potentialen die Aufenthaltswahrscheinlichkeit von einem definierten Atomtyp in einer bestimmten Umgebung der Bindetasche des Zielenzyms vorhersagen. Mit Hilfe der elf Fragmentkristallstrukturen haben wir das Programm HotspotsX validiert. Durch die chemische Diversität und die diversen Posen der Fragmente erhielten wir Hinweise auf aromatische, Donor, Akzeptor, Doneptor und hydrophobe Binder. Die berechneten HotspotsX Karten passen hervorragend zu den experimentell ermittelten Bindungsposen der Fragmente. Das Programm HotspotsX wurde ebenfalls an Kristallstrukturen von Sonden-Molekülen wie Phenol, Harnstoff und Methylharnstoff getestet. Die Bindungsposen dieser Sonden konnten in verschiedenen Zielenzymen mit Hilfe von Röntgenstrukturanalysen ermittelt werden. Die meisten experimentell bestimmten bevorzugten Bindungsregionen passten hervorragend mit den computer-vorhergesagten Positionen überein. Beginnend mit der Sondenstruktur von Phenol in der cAMP abhängigen Protein Kinase A (PKA) haben wir Fragment-basierte Prinzipien angewendet. Sie beruhen auf dem Wachstum von inital entdeckten Fragmenten. In der Kristallstruktur findet man drei Phenolmoleküle, wobei zwei die ATP-Bindestelle besetzen und das andere auf der Glycin-reichen Schleife (G-Schleife) sitzt. Eine computer-basierte Suche wurde durchgeführt. Es wurden Phenol-artige Strukturen vorgeschlagen und für eine konnte eine Kristallstruktur bestimmt werden. Diese Struktur wurde verwendet, um die bevorzugten Bindungsregionen der Bindungstasche auszuleuchten. Das Programm schlug vor, den Liganden in Richtung der G-Schleife zu wachsen, in dieser Region eine aromatische Gruppe zu platzieren und Lys72 mit einer Akzeptorgruppe zu adressieren. Die erste Testverbindung hatte einen Inhibitionswert von 70µM. Im folgenden ersten Design-Zyklus konnte die Affinität auf 6.5µM gesteigert werden. Im zweiten Zyklus wurde eine zusätzliche Aminogruppe an die Leitstruktur synthetisiert, was das Verhältnis Schweratome zu Affinität auf 0.38 anhob. Im letzten Zyklus wurden eine bzw. zwei Methylen-Gruppen als Brücken für die Aminogruppe synthetisiert. Die Affinität für das synthetisierte Diastereomerengemisch zeigte eine Affinität von ca. 110nM. Die Struktur des Phenol-PKA Komplexes zeigte eine mögliche allosterische Bindetasche. Die G-Schleife in diesem Komplex liegt in einer eingeklappten Konformation vor. Diese Konformation könnte durch eines der drei Phenolmoleküle, das auf der G-Schleife sitzt, erzwungen werden. Mehrere Moleküldynamik (MD) Berechnungen wurden durchgeführt, wobei verschiedene Kombinationen bezüglich der Besetzung der drei Phenolmoleküle ausprobiert wurden, um einen Einblick in erste Schritte bei dem Öffnen der Schleife zu bekommen. Die MD Simulation, bei welcher das Phenolmolekül auf der Schleife fehlte, zeigte erste Anzeichen für ein Öffnen der Schleife, was die ersten Schritte eines kaskadenartigen Öffnens darstellen könnte. In einem weiteren Projekt wurde eine virtuelle Suche nach neuen Leitstrukturen der Aldose Reduktase durchgeführt. Von den besten Leitstrukturen wurden fünf Verbindungen synthetisiert und die Affinität gemessen. Unter diesen Leitstrukturen war eine Verbindung, die eine Affinität von 920nM aufwies. Die etablierten Affinitäts-Testsysteme sind, um Fragmente als Binder zu finden, noch nicht ausreichend. Die Herausforderungen liegen in der schwachen Affinität und der schlechten Löslichkeit der Fragmente. Daher wurde der bekannte Temperatur-Stabilitäts-Test auf Fragmente angewendet. Um die Methode zu etablieren, wurden verschiedene Mutanten von EctD charakterisiert. Im letzten Projekt wurden Bindungsposen von Arachidonsäurederivaten in einem K+ Kanal erzeugt, um eine Vorstellung zu bekommen, wie die Bindung aussehen könnte. Eine genaue Bindungspose konnte nicht bestimmt werden, es konnte allerdings gezeigt werden, dass nur ein einzelnes Arachidonsäuremolekül die innere Pore des Kanals blockieren kann.

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