Novel Leads for Selective Antibiotics Against Shigellosis by Virtual Screening, Crystallography and Synthesis

Eubacterial tRNA-guanine transglycosylase (TGT) is involved in the hypermodification of cognate tRNAs leading to an exchange of guanine 34 at the wobble position in the anticodon loop by preQ 1, as part of the biosynthesis of queuine. Mutation of the tgt-gene in Shigella flexneri results in a significant loss of pathogenicity of the bacterium, revealing TGT as a prospective target for the design of potent drugs against Shigellosis. The X-ray structure of Zymomonas mobilis TGT in complex with preQ 1 was used to search for putative inhibitors, initially with the computer program LUDI. Furthermore, the recognition properties of the protein binding site have been used to derive a protein-based pharmacophore which served as a prerequisite for virtual screening based on molecular similarity and docking. This strategy retrieved several novel scaffolds potentially matching with the substrate recognition site. Iterative design has been applied to reveal significantly larger inhibitors with improved binding properties addressing further polar residues in the binding pocket and filling specifically a small hydrophobic cavity. The protein performs several conformational adaptations upon ligand binding which are in agreement with the required substrate promiscuity of the enzyme. Water molecules accommodated in the binding pocket have been detected as important either for mediating interactions between protein and ligand or to bridge interactions between polar groups of the protein. In the latter case, replacement of these waters is detrimental to ligand binding. Addressing the U33 binding pocket reveals a substantial increase in ligand binding affinity, also due to the formation of charge-assisted hydrogen bonds.

Keywords: Shigella Dysentery, Leads Compound Discovery, Drug Discovery and Design, Protein-Ligand Interactions, Docking, Scoring.