Bacterial thioredoxin systems as potential drug targets in novel antimicrobial therapies.

Antimicrobial resistance is a big problem in modern medicine. One of the key components in the combat is the characterization of novel drug targets and the development of new classes of compounds targeting them. In the case of the gastric pathogen Helicobacter pylori, increasing resistance necessitates more elaborate and costly treatment-regimes, which cause more side effects than the standard therapy. As for Bacillus anthracis, the causative agent of anthrax, naturally occurring or engineered resistance threatens to compromise the treatment of infections caused by this severe pathogen. The aim of this thesis was to characterize bacterial thioredoxin systems as potential drug targets in bacteria devoid of glutathione. Furthermore, to develop inhibitors that targets these systems. Special emphasis was given to H. pylori as a representative for slow growing, chronic, pathogens and B. anthracis for rapidly growing pathogens giving rise to fulminant disease. Paper nr I: We provided crystal structures of H. pylori thioredoxin reductase in both the oxidized and reduced forms at 1.7 and 1. 45Å resolution, respectively. This information could potentially be used for rational design and optimization of inhibitors. Paper nr II: We characterized the thioredoxin system of B. anthracis and concluded that, despite an apparent redundancy, B. anthracis has only one thioredoxin reductase, two thioredoxins and one NrdH-redoxin. Furthermore, thioredoxin 1 was the predominant disulfide reductase and the physiologically relevant electron donor for ribonucleotide reductase. This suggests that the thioredoxin system of B. anthracis could be an attractive drug target. Paper III: We described inhibition of bacterial thioredoxin reductases as a novel antibiotic principle using ebselen. Using pure proteins, we could show that ebselen is a competitive inhibitor of E. coli thioredoxin reductase, acting by binding to the C-terminal active site cysteine. We used various E. coli strains with gene-deletions in the redox systems to show that the absence of glutathione confers sensitivity to ebselen. We also showed that H. pylori and M. tuberculosis, both of which naturally lack glutathione, are sensitive to the drug. Manuscript IV: We studied ebselen and derivatives thereof as inhibitors of B. anthracis thioredoxin reductase and tested their antibacterial activity against B. subtilis. We could show that selected compounds are potent inhibitors of B. anthracis thioredoxin reductase with IC50 values down to 70 nM. Minimal Inhibitory Concentrations (MICs) were down to 0.4 μM (0.12μg/ml) for B. subtilis and compounds had a bacteriocidal mode of action. Based on this, we constructed a structure-activity-relationship and concluded that ebselen and derivatives thereof could potentially be used for the treatment of anthrax. In conclusion, the present thesis deals with the characterization of bacterial thioredoxin systems as potential drug targets on a biochemical, bacteriological and structural level. The results obtained suggest that bacterial thioredoxin systems are attractive drug targets in bacteria devoid of glutathione and that ebselen might be a viable starting point for drug development. Inhibitors could potentially be used to treat a wide variety of infections including anthrax, gastric ulcers and tuberculosis. LIST OF PUBLICATIONS I. Gustafsson TN, Sandalova T, Lu J, Holmgren A, Schneider G. Highresolution structures of oxidized and reduced thioredoxin reductase from Helicobacter pylori. Acta Crystallographica section D Biological Crystallography. 2007 Jul;63(Pt 7):833-43. II. Gustafsson TN, Sahlin M, Lu J, Sjöberg BM, Holmgren A. Bacillus anthracis Thioredoxin Systems, Characterization and Role as Electron Donors for Ribonucleotide Reductase. Journal of Biological Chemistry. 2012 Nov 16;287(47):39686-97. III. Lu J, Vlamis-Gardikas A, Kandasamy K, Zhao R, Gustafsson TN, Engstrand L, Hoffner S, Engman L, Holmgren A. Inhibition of bacterial thioredoxin reductase: an antibiotic mechanism targeting bacteria lacking glutathione. FASEB Journal. 2012 Dec 17. [Epub ahead of print] IV. Gustafsson TN, Kandasamy K, Engman L, Holmgren A. Ebselen and analogs as inhibitors of Bacillus anthracis thioredoxin reductase – antibacterial properties with a bacteriocidal mode of action. Manuscript