TGT a Drug Target to Study pKa Shifts, Residual Solvation & Protein-Protein Interface Formation
In this thesis multiple computer aided methods of structure based drug design along with X-ray crystallography and kinetic measurements were used to investigate inhibitors of tRNA-guanine transglycosylase (TGT) a putative target for a new specific antibiotic against Shigella bacteria. Within a year...
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|Summary:||In this thesis multiple computer aided methods of structure based drug design along with X-ray crystallography and kinetic measurements were used to investigate inhibitors of tRNA-guanine transglycosylase (TGT) a putative target for a new specific antibiotic against Shigella bacteria. Within a year about 160 million infections are reported leading to approximately 1 million deaths, predominantly in developing.
The crystallization of Z. mobilis TGT, which offers a nearly identical active site as TGT from S. flexneri, was successfully performed in previous studies. Several scaffolds based on pyridazinones, pteridines, and quinazolinones were discovered with binding affinities in the micro molar range. In addition, a “stretched” guanine with an inserted central six-membered ring, leading to lin-benzoguanine (3 µM), was discovered and further evaluated in this thesis.
During the optimization process of the lin-benzoguanine skeleton two often neglected aspects of ligand binding to a protein are highlighted: (i) pKa shifts are studied once the inhibitor is transferred from aqueous solution to a protein environment and (ii) the influence of residual solvation of amino acids in the active site are investigated during the site chain design of the parent skeleton. The decoration of the lin-benzoguanine with new side chains delivered binding affinities in the low nano molar range (best 2 nM).
In addition, the constitution of the catalytic active complex of TGT and its substrate-tRNA was studied, applying site directed mutagenesis, kinetic measurements and nanoESI-MS experiments. Based on a crystal structure of TGT with a tRNA-stem loop we suggested that TGT is active as a homo dimer and can bind one tRNA molecule for catalysis. The obtained results confirmed that TGT is a homo dimer and binds one tRNA molecule.|