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Structural and Functional Studies of tRNA-Guanine Transglycosylase: A putative Drug Target for Shigellosis Therapy
In this study structure-based drug design approaches for Zymomonas mobilis tRNA - guanine transglycosylase (TGT) are presented. In Shigella sp., the causative agent of bacterial shigellosis, TGT is involved in pathogenicity. Concerning the amino acid sequence, TGTs from both genera are highly h...
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| Format: | Dissertation |
| Sprache: | Englisch |
| Veröffentlicht: |
Philipps-Universität Marburg
2006
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| Zusammenfassung: | In this study structure-based drug design approaches for Zymomonas mobilis tRNA - guanine transglycosylase (TGT) are presented. In Shigella sp., the causative agent of bacterial shigellosis, TGT is involved in pathogenicity. Concerning the amino acid sequence, TGTs from both genera are highly homologous, however, for the Shigella TGT no crystal structures are available. The development of inhibitors for Z. mobilis TGT could assist to develop a potent new antibiotic against shigellosis.
In the first part of this study functional and structural analyses are presented providing valuable information about the base exchange reaction catalyzed by TGT. The comparison of crystal structures from the eubacterial Z. mobilis QueTGT and the archaebacterial Pyrococcus horikoshii ArcTGT, both in complex with tRNA, allowed to develop a detailed model for the base exchange reaction pathway. These analyses indicate an evolutionarily conserved mechanism in all kingdoms of life. Additionally, the tRNA bound TGT crystal structures also allow to classify the TGT superfamily into cluster of superfamilies with TIM-barrel fold sharing a standard phosphate binding motif.
In QueTGT crystal structures from two species conserved TGT dimers were observed. Their similarity along with additional data of TGT amino acid sequences from 21 species indicates functional dimer formation in eubacterial and eukaryotic QueTGTs.
To support the base exchange reaction in QueTGT the binding pocket has to perform a structural and functional rearrangement controlled by the general acid/base Glu235. However, this adaptability allows substrate promiscuity. In addition to the natural substrate preQ1 also incorporation of its biosynthetic precursor preQ0 is possible. A mutational study, exchanging Glu235 against Gln, resulted in the inversion of selectivity in favour of preQ0. Nevertheless, crystal structure analyses revealed that the preQ0 selective binding pocket of the mutant is stabilized by unexpected interactions.
The results from the structural and functional analyses of TGT were implemented in three structure-based inhibitor design campaigns presented in the second part of this study. All tested compounds were provided by cooperation partners. In a first step the available test system had to be modified to reflect previously unconsidered competitive and uncompetitive inhibitory contributions. Therefore inhibition constants of already published compounds were redetermined to revalidate the structure affinity relationship.
In the first and most successful campaign lin-benzoguanine was established as new lead structure. Crystal structure analyses revealed, that lin-benzoguanines induce conformational changes in the binding pocket extending its size. Similar changes are also observed upon binding of tRNA to TGT. Water molecules were identified to be involved in this rearrangement procedure. 2- and 4-substituted lin-benzoguanines enabled the factorization of favourable and unfavourable contributions towards binding. 4-Substituted derivatives disturb a water network adjacent to the negatively charged nucleophile Asp280 resulting in a slight affinity loss. 2-substituted derivatives exhibit nanomolar inhibition and gain affinity by a factor of 10 compared to lin-benzoguanine. Charge assisted binding resulting from the 2-substitution seems to be the key factor for the significant affinity gain.
In the second approach a compound identified in a previous virtual screening campaign was optimized. For this compound an unusual binding mode was predicted, however, crystallization in complex with TGT was not successful. 1- and 5-substituted derivatives of benzimidazolin-2-one/-thione were tested exhibiting 2 – 150 µM inhibition. Crystal structure analyses of these compounds in complex with TGT were not successful. Docking experiments provide no conclusive picture about the binding mode. Thus, structure – affinity relationships still remains to be resolved.
In a third approach apigenin-based compounds isolated from plant extracts by “ligand fishing” were tested kinetically and crystallographically. Inhibition constants are in the range of 20 – 90 µM. Unfortunately, crystallization in complex with TGT was not successful. Docking studies do not suggest a fully convincing specific recognition of these compounds in the active site of TGT. Therefore, the exact binding mode still remains to be elucidated. |
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| Umfang: | 173 Seiten |
| DOI: | 10.17192/z2006.0562 |