Studies on Human and Drosophila melanogaster Glutathione Transfer- ases of Biomedical and Biotechno- logical Interest

Parts of this thesis are based on a previous licentiate thesis v To the martyrs of APS attack, Peshawar vi vii Identification of new inhibitors for human hematopoietic prostaglandin D2 synthase among FDA-approved drugs and other compounds, Chem Biol Interact 229 (2015) 91-99. Overexpression of glutathione transferase E7 in Drosophila differentially impacts toxicity of organic isothiocyanates in males and females, PloS One 9 (2014) e110103. Drosophila GSTs display outstanding catalytic efficiencies with the environmental pollutants 2,4,6-trinitrotoluene and 2,4-dinitrotoluene, Biochem Biophys Rep 5 (2016) 141-145. Comparison of epsilon-and delta-class glutathione S-transferases: the crystal structures of the glutathione S-transferases DmGSTE6 and DmGSTE7 from Drosophila melanogaster, Acta Cryst D Biol Cryst 71 (2015) 2089-2098. Abstract Glutathione transferases (GSTs, EC.2.5.1.18) are multifunctional enzymes that are universally distributed in all cellular life forms. They play important roles in metabolism and detoxication of endogenously produced toxic compounds and xenobiotics. GSTs have gained considerable interest over the years for biomedical and biotechnological applications due to their involvement in the conjugation of glutathione (GSH) to a vast array of chemical species. Additionally, the emergence of non-detoxifying functions of GSTs has further increased their biological significance. The present work encompasses four scientific studies aimed at investigating human as well as fruit fly Drosophila melanogaster GSTs. Paper I presents the immobilization of GSTs on nanoporous alumina membranes. Kinetic analyses with 1-chloro-2,4-dinitrobenzene followed by specificity screening with alternative substrates showed a good correlation between the data obtained from immobilized enzymes and the enzymes in solution. Furthermore, immobilization showed no adverse effects on the stability of the enzymes. Paper II presents inhibition studies of human hematopoietic prostaglandin D2 synthase (HPGDS), a promising therapeutic target for anti-allergic and anti-in-flammatory drugs. Our screening results with an FDA-approved drug library revealed a number of effective inhibitors of HPGDS with IC50 values in the low micromolar range. Paper III concerns the toxicity of organic isothiocyanates (ITCs) that showed high catalytic activities with GSTE7 in vitro. The in vivo results showed that phenethyl isothi-ocyanate (PEITC) and allyl isothiocyanate in millimolar dietary concentrations conferred toxicity to the adult fruit flies leading to death or shortened lifespan. The transgenic female flies overexpressing GSTE7 showed increased tolerance against PEITC toxicity compared to the wild-type. However, the effect was opposite in male flies overexpress-ing GSTE7 after one week exposure. Notably, the transgene enhanced the oviposition activity of flies with and without ITCs exposure. Paper IV highlights Drosophila GSTs as efficient catalysts …