water cluster analysis In this thesis different methods of structure-based drug design have been applied to develop TGT inhibitors suiting the manifold purposes of the tackled projects. TGT is not only a putative target for the treatment of Shigellosis, but additionally represents a well-accessible model system to study more general aspects of drug design. The presented results are mostly received from X-ray crystallography and binding affinity measurements. For the latter determinations, a new method - microscale thermophoresis - has been introduced. To characterize binding of TGT ligands, the newly established protocol largely replaces the expensive and time-consuming kinetic assay based on radioactive labeled guanine. The experimental perspective has been further expanded by a point mutation study, methods of computational chemistry and non-covalent nanoESI mass spectrometry. The latter technique provided crucial information about the dimer stability of TGT in solution and upon the impact of ligands, designed to perturb the anticipated hot spot interactions of the dimer interface (section 2). For the first time, a dimer destabilization in solution has been verified induced by needle-type decorated ligands which spike into the interface region. However, these ligands were not able to fully disrupt the dimer which questions the importance of the targeted substructures for dimer stability. A detailed crystal structure analysis of three TGT-ligand complexes complemented with collected affinity data could unravel the role of the targeted helix-loop-substructure, assumed to be crucial for dimer stability due to preceding mutation studies. Despite the involvement of this substructure in several, crystallographically conserved directed interactions between the monomer mates this helix-loop-motif is most likely flexible in solution and just conveyed into a well-ordered arrangement upon crystallization. Supported by molecular modeling and MD-simulations it was possible to provide an explanation for the discrepancy between the discovered minor importance of these helix-loop associated directed interactions and the observed ligand-induced dimer destabilization in solution. In section 3 two series of TGT inhibitors have been developed - each corresponding to another variation of the lin-benzoguanine scaffold. The lin-benzohypoxanthines - lacking the exocyclic amino function at C(6) - and the C(6)-N-alkylated lin-benzoguanines have been analyzed with respect to their membrane permeability, pKa-profile, binding mode in crystal structures and binding affinity. Finally these characteristics have been compared to the parental lin-benzoguanines studied in previous works. For the pyrimidine portion of lin-benzohypoxanthines a shift of ca. two pKa units to increased acidity could be achieved compared to the analog lin-benzoguanines. However, despite the reduced tendency to be charged, lin-benzohypoxanthines showed no improved membrane permeability. The lin-benzohypoxanthines exhibit strongly reduced binding affinities in the micromolar range which could be reasoned by the loss of two H-bonds formed between the exocyclic amino function of parental lin-benzoguanines and two aspartates of the binding pocket. A water cluster was identified as a crystallographically conserved arrangement being picked up upon binding of lin-benzohypoxanthines. This cluster experiences several favorable interactions to the protein and to the ligand and was partially found also in the apo structure. Despite this water cluster cannot compensate the loss in affinity for lin-benzohypoxanthines, the potential large impact of favorable water-protein interactions on the ligand´s binding affinity could be demonstrated by crystal structure analysis of two C(6)-N-alkylated lin-benzoguanines. In section 4, a series of disubstituted lin-benzoguanines and lin-benzohypoxanthines has been characterized in terms of their binding mode and affinity. The combination of substituents simultaneously targeting the ribose33- and ribose34-pocket yielded the first TGT inhibitor exhibiting picomolar binding affinity. However, the comparison of disubstituted compounds with their monosubstituted analogs revealed that the overall binding affinity is not necessarily improved by sidechain combination to result in additivity of affinity contributions. The results presented in section 2 concerning the flexibility of a dimer interface-associated helix-loop substructure also affect the interpretation of crystal structures in section 4. In section 4, a more rigid, crystallographically defined binding mode within the ribose33-pocket could be obtained for the C(2)-substituents of three compounds of the analyzed series. In section 5, a mutationally introduced asparagine within the TGT recognition pocket can be used as a model for a permanently protonated aspartate, which is involved in ligand binding and catalysis. lin-Benzopurine als Inhibitoren der tRNA-Guanin Transglycosylase: Störung der Homodimer Formation, Import von Wassernetzwerken und Determinanten kristallographischer Unordnung Im Rahmen dieser Dissertation wurden Inhibitoren der TGT unter verschiedenen Gesichtspunkten entwickelt und untersucht. Hierbei kamen zahlreiche Methoden des strukturbasierten Wirkstoffdesign zum Einsatz. Die TGT-Dimerstabilität in Lösung und deren Beeinflussbarkeit durch Liganden wurde untersucht (Kapitel 2). Erstmals konnte eine ligandinduzierte Destabilisierung des Homodimers in Lösung nachgewiesen werden, welche auf die nadelartig in die interface-Region reichenden Ligandseitenketten zurückgeführt werden konnte. Die detaillierte Analyse von drei TGT-Ligand-Komplexstrukturen, komplementiert mit entsprechenden Affinitätsdaten, konnte den Einfluss der angesteuerten Helix-loop-Substruktur auf die Dimerstabilität offenlegen. War diese aufgrund vorausgegangener Mutationsstudien noch als entscheidend für den Zusammenhalt der assemblierten TGT-Monomere eingeschätzt worden, ist das Strukturelement, trotz seiner Beteiligung an zahlreichen kristallographisch konservierten Wechselwirkungen zum korrespondierenden Monomer, von nur geringer Bedeutung für die Dimerstabilität. Die sich ergebende Widersprüchlichkeit zwischen der ligandinduzierten Dimerdestabilisierung in Lösung und der offensichtlich geringen Bedeutung der Helix-loop-assoziierten gerichteten Wechselwirkungen konnte durch molecular modelling und Molekulardynamik-Simulationen aufgeklärt werden. Die kristallstrukturbasierten Simulationen zeigten, dass die beschriebene loop-Struktur - auch in Gegenwart eines gebundenen, nadelartig dekorierten Liganden - einer hydrophoben Kontaktfläche zwischen beiden Monomeren deckelartig aufliegt. Eine hydrophobe Triade bestehend aus drei aromatischen Resten ist an der Bildung dieser Kontaktfläche beteiligt und konnte aufgrund ihrer Konservierung in allen untersuchten Ligandkomplexen als wahrscheinliche hot spot-Interaktion des Dimer interface ausgemacht werden. In Kapitel 3 wurden zwei Serien von TGT Inhibitoren entwickelt und untersucht. Jede Serie basiert auf einer Variation des in vorangegangenen Arbeiten entwickelten lin-Benzoguanin-Grundgerüsts. Die lin-Benzohypoxanthine, bei denen die exozyklische Aminofunktion an C(6) des Trizyklus entfernt wurde, und die C(6)-N-alkylierten lin-Benzoguanine wurden hinsichtlich Membranpermeabilität, pKa-Profil, Bindungsmodus in Kristallstrukturen und Bindungsaffinität charakterisiert. Die festgestellten Eigenschaften wurden mit den analogen lin-Benzoguaninen verglichen. Für die Pyrimidin-Partialstruktur der lin-Benzohypoxanthine konnte eine Verschiebung von zwei pKa-Einheiten zu erhöhter Azidität gegenüber den analogen lin-Benzoguaninen erzielt werden. Trotz verminderter Protonierbarkeit der lin-Benzohypoxanthine konnte aber keine verbesserte Membranpermeabilität erreicht werden. Sehr wahrscheinlich wird ein günstigeres biopharmazeutisches Profil durch die in allen untersuchten Verbindungen erhaltene, leicht protonierbare 2-Aminoimidazol-Partialstruktur verhindert. Ein Wassernetzwerk konnte als ein mit der Bindung von lin-Benzohypoxanthinen assoziiertes, kristallographisch konserviertes Arrangement dargestellt werden. Das teilweise auch in der apo-Struktur sichtbare Netzwerk bildet günstige Wechselwirkungen sowohl zu gebundenen lin-Benzohypoxanthinen als auch zu Resten der Bindetasche aus. Obgleich das Netzwerk den Affinitätsverlust gegenüber den lin-Benzoguaninen nicht kompensieren kann, konnte anhand der Komplexstrukturen zweier C(6)-N-alkylierter lin-Benzoguanine der potentiell große Einfluss günstiger Protein-Wasser- und Ligand-Wasser-Interaktionen auf die Bindungs-affinität gezeigt werden. In Kapitel 4 wurde eine Serie disubstituierter lin-Benzoguanine und lin-Benzohypoxanthine im Hinblick auf den kristallographischen Bindungsmodus und die Bindungsaffinität charakterisiert. Die Kombination zweier Substituenten am trizyklischen Grundgerüst ermöglicht die zeitgleiche Bindung der Liganden in der Ribose33- und Ribose34-Tasche. Hierdurch konnte erstmals eine pikomolare Bindungsaffinität für einen TGT-Inhibitor erreicht werden. Der Vergleich der beschriebenen disubstituierten Verbindungen mit ihren monosubstituierten Analoga machte jedoch deutlich, dass sich die Beiträge der einzelnen Substituenten zur Bindungsaffinität nicht zwingend additiv verhalten. Weiterhin konnte in Kapitel 4 erstmals ein kristallographisch geordneter Bindungsmodus für die C(2)-Seitenketten dreier Liganden in der Ribose33-Tasche festgestellt werden. Auffällig ist hierbei die Ummantelung der apolaren Seitenketten durch kristallographisch definierte Wassernetzwerke, welche die geordneten Konformationen vermutlich stabilisieren. Ein signifikanter Einfluss des geordneteren Bindungsmodus auf die Bindungsaffinität der entsprechenden Liganden ist hingegen nicht festzustellen. Kapitel 5 beschreibt eine TGT-Variante, in deren Bindetasche ein Aspartat mittels positionsgerichteter Mutagenese durch ein Asparagin ersetzt wurde. ths Prof. Dr. Klebe Gerhard Klebe, Gerhard (Prof. Dr.) 2013-01-30 https://doi.org/10.17192/z2013.0074 opus:4713 lin-Benzopurines as Inhibitors of tRNA-Guanine Transglycosylase: Perturbance of Homodimer Formation, Import of Water Clusters and Determinants of Crystallographical Disorder Inhibitordesign Protein Protein Interaktion 2015-05-12 Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg Investigations on lin-Benzopurines With Respect to Dissociation Behavior, Pocket Cross-Talk, Targeting Resistance Mutants, Residual Mobility, and Scaffold Optimization https://archiv.ub.uni-marburg.de/diss/z2013/0074/cover.png Philipps-Universität Marburg Wassernetzwerkanalyse application/pdf Enzyminhibitor Immekus, Florian Peter Philip Immekus Florian Peter Philip English Sansonetti, P. J. (2001). III. 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Proteinkristallographie 2013-02-11 2012 protein protein interaction Fachbereich Pharmazie monograph doctoralThesis Queuin-tRNS-Ribosyltransferase urn:nbn:de:hebis:04-z2013-00742 inhibitor design Natural sciences + mathematics Naturwissenschaften Pharmazeutische Chemie