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 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-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 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

Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn) by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering) by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate

High-affinity inhibitors of Zymomonas mobilis tRNA-guanine transglycosylase through convergent optimization.

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Crystallographic data collection and refinement statistics – Tgt(Cys158Val/Val233Gly), “WT”.

<p>Crystallographic data collection and refinement statistics – Tgt(Cys158Val/Val233Gly), “WT”.</p

Substrate base binding pocket of <i>Z.</i> <i>mobilis</i> Tgt and modelled human Tgt.<b> </b>

<p>A) Detail of <i>Z. mobilis</i> Tgt·preQ₁ complex crystal structure (PDB-code: <b><u>1p0e</u></b>) showing the active site with the bound substrate in stick representation. Carbon atoms of protein residues are coloured in green, those of preQ₁ in orange. B) Homology model of human Tgt created with the <i>Z. mobilis</i> Tgt crystal structure as a template. The close up shows active site residues (carbon atoms in grey) superimposed with preQ₁ (carbon atoms in orange) as present in <b><u>1p0e</u></b>. The coordinates of the homology model are provided within the Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064240#pone.0064240.s001" target="_blank">Coordinates S1</a>).</p

Trapping experiments performed with Tgt/tRNA mixtures in presence of queuine or 2,6-diamino-3<i>H</i>-quinazolin-4-one.

<p>A) Chemical structure of 2,6-diamino-3<i>H</i>-quinazolin-4-one (DAQ), an uncompetitive inhibitor of Tgt. B) SDS-PAGE analysis of reaction mixtures of Tgt or mutated variants thereof and tRNA^Tyr under conditions indicated. SM, size marker; q, queuine. While DAQ causes retarded Tgt bands by stabilising the covalent Tgt·tRNA intermediate, queuine lacks this ability for most of the investigated Tgt variants. Solely in case of Tgt(Cys159Val/Val233Gly) a faint retarded band is visible indicating that queuine may to some extent be able to bind to the guanine 34/preQ₁ subpocket of the covalent enzyme·tRNA complex.</p

Crystallographic data collection and refinement statistics - Tgt(Cys158Val).

<p>Crystallographic data collection and refinement statistics - Tgt(Cys158Val).</p

Launching Spiking Ligands into a Protein–Protein Interface: A Promising Strategy To Destabilize and Break Interface Formation in a tRNA Modifying Enzyme

Apart from competitive active-site inhibition of protein function, perturbance of protein–protein interactions by small molecules in oligodomain enzymes opens new perspectives for innovative therapeutics. tRNA–guanine transglycosylase (TGT), a potential target to treat shigellosis, is active only as the homodimer. Consequently, disruption of the dimer interface by small molecules provides a novel inhibition mode. A special feature of this enzyme is the short distance between active site and rim of the dimer interface. This suggests design of expanded active-site inhibitors decorated with rigid, needle-type substituents to spike into potential hot spots of the interaction interface. Ligands with attached ethinyl-type substituents have been synthesized and characterized by <i>K</i>_d measurements, crystallography, noncovalent mass spectrometry, and computer simulations. In contrast to previously determined crystal structures with nonextended active-site inhibitors, a well-defined loop-helix motif, involved in several contacts across the dimer interface, falls apart and suggests enhanced flexibility once the spiking ligands are bound. Mass spectrometry indicates significant destabilization but not full disruption of the complexed TGT homodimer in solution. As directed interactions of the loop-helix motif obviously do not determine dimer stability, a structurally conserved hydrophobic patch composed of several aromatic amino acids is suggested as interaction hot spot. The residues of this patch reside on a structurally highly conserved helix-turn-helix motif, which remains unaffected by the bound spiking ligands. Nevertheless, it is shielded from solvent access by the loop-helix motif that becomes perturbed upon binding of the spiking ligands, which serves as a possible explanation for reduced interface stability

Superimposition of preQ₁-bound Tgt(Cys158Val) with its apo-, its guanine- and its queuine-bound form.

<p>Carbon atoms of the Tgt(Cys158Val)·preQ₁ complex are coloured pink in all sub-figures. (A) Superimposition (based on c_α) of preQ₁-bound Tgt(Cys158Val) and apo-Tgt(Cys158Val). Carbon atoms of apo-Tgt(Cys158Val) are coloured blue. Binding of preQ₁ to Tgt(Cys158Val) provokes a shift of Val158 towards the ligand leading to the displacement of two water molecules (W1 and W2; shown as red spheres) which are present within this region in apo-Tgt(Cys158Val) and absent in the complex structure. In addition, the side chain of the proximate Thr159 rotates by about 90°. Exactly the same structural changes upon binding of preQ₁ are observed for Tgt(Cys158Val/Val233Gly). (B) Superimposition (based on c_α) of preQ₁-bound Tgt(Cys158Val) and guanine-bound Tgt(Cys158Val). Carbon atoms of the Tgt(Cys158Val)·guanine complex are coloured yellow. In the Tgt(Cys158Val)·guanine complex, the side chain of Val158 becomes largely disordered. The Thr159 side chain adopts a similar conformation as observed in the apo-structure. (C) Superimposition (based on c_α) of preQ₁-bound Tgt(Cys158Val) and queuine-bound Tgt(Cys158Val). Carbon atoms of the Tgt(Cys158Val)·queuine complex are shown in grey. Binding of queuine obviously leads to disordering of the Val158 side chain as no electron density attributable to this isopropyl moiety is present in the electron density map of the refined Tgt(Cys158Val)·queuine complex structure. Also upon binding of queuine no conformational change of the Thr159 side chain is observed. It adopts a similar conformation as in the apo- and guanine-bound structures.</p