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Titel:Strukturbasierte Entwicklung und Charakterisierung von Inhibitoren der TGT,ein mögliches Ziel zur Therapie der Bakterienruhr
Autor:Ehrmann, Frederik Rainer
Weitere Beteiligte: Klebe, Gerhard (Prof.Dr.)
Veröffentlicht:2016
URI:https://archiv.ub.uni-marburg.de/diss/z2017/0064
DOI: https://doi.org/10.17192/z2017.0064
URN: urn:nbn:de:hebis:04-z2017-00648
DDC: Allgemeines, Wissenschaft
Titel(trans.):Structure based design and characterization of TGT inhibitors, a putative target for therapy of Shigellosis
Publikationsdatum:2017-02-07
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Proteine, Shigellosis, Kristallisation, TGT, Inhibitor, Inhibitor, Homodimer, Homodimer, Bakterienruhr, Arzneimitteldesign, Drug design

Zusammenfassung:
Das Zielenzym TGT hat eine essentielle Bedeutung für die Pathogenese der entzündlichen Darmerkrankung Bakterienruhr, die durch Shigellen verursacht wird. Die TGT tauscht dazu die Purinbase Guanin in der Wobble Position-34 von verschiedenen tRNAs durch die modifizierte Base preQ1 aus. Die von den Purinbasen abgeleiteten lin-Benzoguanin Inhibitoren binden im aktiven Zentrum der Basenaustauschreaktion. Durch Anfügen von Substituenten können die benachbarten Ribose-33 und Ribose-34-Taschen adressiert werden. In zwei Studien wurden die Wassernetzwerke der genannten Seitentaschen ladungsneutral durch Monosaccharid-Substituenten der lin-Benzoguanin Inhibitoren adressiert und verdrängt. Dabei binden die Furanosyl-Substituenten räumlich gesehen in einer vergleichbaren Region wie die Phosphatgruppe-35 des natürlichen tRNA-Substrats. Dies lässt den Rückschluss zu, dass Furanoside als mögliche Surrogate für die Adressierung von Phosphatbindetaschen geeignet sind. Da die TGT nur als Homodimer das natürliche tRNA-Substrat modifiziert, kommen als Inhibitionsmechanismen sowohl die Blockierung des aktiven Zentrums, als auch die Störung der dimeren Quartärstruktur der TGT, in Frage. Die Kontaktfläche der zwei TGT Monomere liegt in unmittelbarer Nähe zu der Ribose-34-Tasche. Bei der Analyse der Kokristallstrukturen der Furanosyl-substituierten Inhibitoren fiel auf, dass durch Adressierung des hydrophoben Bereichs der Seitentasche ein angrenzendes Loop-Helix Motiv in seiner geometrischen Integrität gestört wird. Da dieses Motiv entscheidend zur Dimerstabilität beiträgt und die Umlagerungen nur in Kokristallstrukturen auftreten, wurden in einer vertieften Studie bereits charakterisierte Liganden kokristallisiert, von denen zuvor nur eine durch die Soaking-Methode erhaltene Kristallstruktur vorlag. Die Analyse dieser Kokristallstrukturen offenbarte zum einen andere Bindungsmodi als in den Soaking-Kristallstrukturen und bestätigte zum anderen weitere Liganden, die das Interface der TGT stören. Die Dimer-destabilisierende Wirkung dieser Liganden wurde durch native Massenspektroskopie bestätigt. Ein Ligand induziert durch seine Bindung eine neuartige Ausrichtung des TGT-Dimers (verdrehtes Dimer), welche offensichtlich aufgrund der verdrehten Neuausrichtung nicht mehr katalytisch aktiv sein kann. In einer Folgestudie konnten drei Inhibitoren charakterisiert werden, die ebenfalls die Dimerisierung der TGT stören. Zwei dieser Verbindungen kokristallisieren sowohl in dem konventionellen, als auch in dem verdrehten Dimer. Eine weitere Studie verfolgte den Prodrug-Ansatz. Da die lin-Benzoguanin-Inhibitoren nicht membrangängig sind, wurde das Grundgerüst mit unterschiedlichen Carbamat-Substituenten versehen. Infolge konnten PAMPA-Messungen zum ersten Mal membrangängige Liganden der TGT bestätigen. In einer Art retrospektiven Arbeit wurden die Bindungsbeiträge der Aminosäuren der Guanin 34/preQ1 Tasche auf die Affinitäten der untersuchten Liganden aufgeklärt. Anhand von Benzimidazol-Derivaten wurde eine zuvor durch eine MD-Simulation prognostizierte Taschenerweiterung erstmals in Kokristallstrukturen experimentell bestätigt. Sowohl Hydrazid-, als auch Guanidin-Substituenten des Benzimidazol-Grundkörpers, stabilisieren aufgrund ihres sterischen Drucks auf Asp156 und der Solvatisierung von Gln203 und Gly230 die transiente Taschenerweiterung. In zukünftigen Designzyklen könnten die Guanidin-Benzimidazole derivatisiert werden, um den neuen Hohlraum zu adressieren und so neue Affinitätspotentiale zu nutzen. Ein weiteres Projekt verfolgte die Charakterisierung eines Nukleosidderivats, welches als mögliches Übergangszustand-Analogon die Basenaustauschreaktion der TGT inhibiert. Der entwickelte Deazaguanin-basierte Inhibitor stellt eine neue Verbindungsklasse und mögliche Leitstruktur dar, die in Zukunft die etablierten lin-Benzoguanine ablösen könnte. In einem größer angelegten Projekt wurde von neun Kristallographen der AG Klebe die in-house Fragmentbibliothek durch Soaking-Kristallstrukturen an das Zielenzym Endothiapepsin röntgenkristallographisch durchgemustert. Von 361 Soaking Kristallstrukturen wurden 71 Komplexstrukturen (Trefferquote 20%) erhalten. Im Zuge dieses Projektes wurde auch ein standardisiertes Verfeinerungsprotokoll entwickelt, welches die Identifizierung von Fragmenten in Kristallstrukturen optimiert und automatisiert. Aufgrund der strukturellen Information der Fragmente in den Kristallstrukturen konnten zum einen neue funktionelle Gruppen zur Adressierung der katalytischen Diade, als auch drei Hotspots abseits des aktiven Zentrums, identifiziert werden. Neben der Demaskierung eines falsch-positiven Treffers aus biophysikalischen Screening-Verfahren, wurde auch offensichtlich, dass die in der Praxis häufig verwendeten biophysikalischen Methoden zum Vor-Screening von Fragmentbibliotheken die Anzahl der Treffer und somit mögliche Röntgenkomplexstrukturen nicht unbedingt erhöht sondern gegebenenfalls reduziert.

Summary:
The target enzyme TGT is essential for the molecular pathogenesis of the inflammatory disease Shigellosis, which is caused by Shigella spp. Incorporation of modified nucleobases into tRNA are required to develop pathogenicity of Shigella. Therefore, TGT replaces the purine base guanine in the wobble position-34 of tRNAsAsn,Asp,His,Tyr by the modified base preQ1. The lin-benzoguanine inhibitors derived from purine bases bind in the active site of the base exchange reaction. By attaching substituents, the adjacent specificity pockets ribose 33 and ribose 34 can be addressed, respectively. In two studies, the water clusters of the named pockets were addressed and displaced by charge neutral monosaccharide substituents of the lin-benzoguanine inhibitors. Thereby, the furanosyl-substituents bind in comparable region as the phosphate group-35 of the natural tRNA substrate. This implies that furanosides might be a surrogate to target phosphate binding sites. As TGT modifies the natural tRNA substrate only as a homodimer, both, blocking of the active site and disturbing the dimeric quaternary structure of the TGT can be considered as inhibition mechanisms. The contact surface of the two TGT monomers is in close proximity to the ribose-34 pocket. Upon analyses of the co-crystal structures of the furanosyl-substituted inhibitors it became obvious, that by addressing the hydrophobic region of the side pocket, an adjacent loop helix motif was disturbed and lost its geometric integrity. This disturbance is associated with rearrangements of up to 20 amino acids. As this motif decisively contributes to the dimeric stability and rearrangements are only visible in co crystal structures, previously characterized ligands have been newly co-crystallized in an elaborate study, as only crystal structures obtained by the soaking method were available. The analysis of these co crystal structures revealed on the one hand different binding poses compared to the soaking structures and on the other hand confirmed further ligands that interfere with TGT interface. The dimer-disturbing effect of these ligands has been confirmed by native mass spectroscopy. One ligand caused a complete rearrangement of TGT monomer units (twisted dimer), leading to an arrangement obviously no longer catalytical active with twisted subunit packing. In a follow-up study, three inhibitors could be characterized, which also interfere with the dimerization of TGT. Two of these compounds co crystallized in both, the conventional and the twisted dimer, respectively. Another study followed a prodrug approach. As the lin-benzoguanine inhibitors do not diffuse through membranes, the scaffold was modified with different carbamate substituents. As a result, PAMPA measurements confirmed ligands of TGT that are membrane-permeable for the first time. Furthermore, from two carbamate derivatives co-crystal structures could be obtained, which due to surprising orientation of the substituents address the ribose-34/phosphate-35 pocket. In a kind of retrospective work the impact of amino acids flanking the guanine-34/preQ1 site on binding affinity of the examined ligands were studied. Based on benzimidazole derivatives, a cavity expansion, previously predicted by a MD-simulation, has been experimentally confirmed by co crystal structures for the first time. Both, hydrazide and guanidine groups attached to the benzimidazole scaffold stabilize a transient cavity opening, due to steric impact on Asp156 and solvation of Gln203 and Gly230. In future design cycles, guanidine-benzimidazoles can be derivatized to address the new pocket and thus exploit new affinity options. Another project followed the characterization of a nucleoside derivative, which inhibits as a putative transition state analogue the base exchange reaction of TGT. The developed deazaguanine-type inhibitor represents a new class of compounds and suggests a possible lead structure that could replace the established lin-benzoguanines in the future. In a large-scale project, nine crystallographers from the group of Prof. Klebe scanned the in-house fragment library by soaking crystal structures of the target enzyme Endothiapepsin. From 361 soaking crystal structures 71 complex structures (hit rate 20%) were obtained. Within the framework of this project, also a standardized refinement-protocol was developed, which optimizes and automatizes the identification of fragments in crystal structures. Based on the structural information of the fragments in the crystal structures new functional groups to address the catalytic dyad, as well as three hotspots remote from the active site were identified. In addition to unmasking false-positive hit from biophysical screening methods, it became obvious that biophysical methods frequently used to initially screen fragment libraries possibly reduces the number of accessible hits and thus the number of X-ray complex structures.

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