lin-Benzopurines as Inhibitors of tRNA-Guanine Transglycosylase: Perturbance of Homodimer Formation, Import of Water Clusters and Determinants of Crystallographical Disorder
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 m...
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|Summary:||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.|