Microsomal prostaglandin E synthase 1 and microsomal glutathione transferase 1 : inhibition and mechanism
The Membrane-Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) is a family of six membrane bound proteins with different biological functions. MAPEG superfamily members enable and catalyze reactions where reactive lipid intermediates are either transformed to physiological messengers or turned into unreactive compounds. Five of these proteins are enzymes that utilize glutathione in their catalytic mechanism, and two of these are the focus of this thesis.
Microsomal prostaglandin E synthase 1 (MPGES1) is induced by pro-inflammatory stimuli and is the major contributor to prostaglandin E2(PGE2) synthesis during inflammation. PGE2 mediates a number of biological responses, including the cardinal signs of inflammation by modulating vasodilation and thereby increasing blood flow, redness and swelling, as well as pain and fever. Increased biosynthesis of PGE2by MPGES1 has been implicated in numerous chronic inflammatory pathologies like rheumatoidarthritis and cancer. MPGES1 is therefore considered a potential drug target and has been investigated by both pharmaceutical companies and academic researchers. In collaboration with a small research and development company, we have developed inhibitors targeting MPGES1 and investigated their inhibitory mechanisms. To do that, we developed a simple medium- to high-throughput assay and concluded that these inhibitors, as well as the reference inhibitor MK-886, function mainly as competitive inhibitors towards its substrate PGH2. We further investigated the inhibitors in rat enzyme and through site directedmutagenesis found that the cleft where the inhibitors enter, and presumably also PGH2enters, has more steric hindrancein the rat enzyme compared to the human enzyme. This discovery enlightened the reason why several potent human MPGES1 inhibitors didn’t have any effect in rodent animal models. Furthermore, we developed dual MPGES1 inhibitors for human and rat enzyme and characterized those in in vitroassays and in in vivorodent models of inflammation. In vivowe compared the prostanoid profile after pharmacological inhibition to that after genetic deletion of MPGES1. Differences in the effect ofMPGES1 pharmacological inhibition and genetic deletion were detected, as well as differences between different models. We conclude thereby that it is important to compare different inhibitors in the samemodels with similar conditions, in order to have a significant comparison, and to complement results from knockout animals with inhibition results in wild type systems.
Microsomal glutathione transferase 1 (MGST1) is a membrane bound glutathione transferase that is involved in cellular protection from oxidative stress and xenobiotics. MGST1 catalyzes conjugation reactions of glutathione to reactive (electrophilic substrates) so they can be more readily excreted from the cells. The enzyme displays broad substrate specificity and is most prevalent in the liver, which is the place where the most important partof drug metabolism occurs. A unique and very interesting feature of this glutathione transferase is that it can be activated by modification with sulfhydryl reagents and proteolysis. Our group has conducted extensive research on MGST1 and uncovered several aspects of its catalytic mechanism through both steady-state kinetics and pre steady-state kinetic experiments. We have now concluded all the information about the microscopic steps of the enzyme’s global kinetic mechanism and derived the steady-state rate expression. By comparing calculated catalytic constants to our experimental values we have discovered a pre-existing resting state to this enzyme which is most pronounced at low temperatures and low reactive electrophilic substrates. We propose that limited turnover pre steady state experiments are the best way to understand the physiologically relevant catalytic mechanism of MGST1.
In conclusion, this thesis provides a deeper understanding of two important members of the MAPEG superfamily of enzymes with different physiological functions and catalytic mechanisms of action. We have gained insights into the structure and inhibitory mechanisms of MPGES1 as well as characterized potent inhibitors of this enzyme in models of inflammation. Our findings constitute new tools in the study of MPGES1. We have also unraveled the global mechanism of MGST1 which is the closest MAPEG member to MPGES1 based on sequence similarity. Our methods used for the determination of the catalytic mechanism of MSGT1 have the potential to assist in defining the catalytic mechanism of MPGES1.
List of scientific papers
I. Identification of Key Residues Determining Species Differences in Inhibitor Binding of Microsomal Prostaglandin E Synthase-1. Pawelzik SC, Uda NR, Spahiu L, Jegerschöld C, Stenberg P, Hebert H, Morgenstern R, Jakobsson PJ. J Biol Chem. 2010; 285 (38): 29254-61
https://doi.org/10.1074/jbc.M110.114454
II. A Facilitated Approach to Evaluate the Inhibitor Mode and Potency of Compounds Targeting Microsomal Prostaglandin E Synthase-1. Spahiu L, Stenberg P, Larsson C, Wannberg J, Alterman M, Kull B, Nekhotiaeva N, Morgenstern R. Assay Drug Dev Technol. 2011; 9 (5): 487-95
https://doi.org/10.1089/adt.2010.0350
III. Characterization of a Human and Murine mPGES-1 Inhibitor and Comparison to mPGES-1 Genetic Deletion in Mouse Models of Inflammation. Leclerc P, Idborg H, Spahiu L, Larsson C, Nekhotiaeva N, Wannberg J, Stenberg P, Korotkova M, Jakobsson PJ. Prostaglandins Other Lipid Mediat. 2013; 107: 26-34
https://doi.org/10.1016/j.prostaglandins.2013.09.001
IV. Global Kinetic Mechanism of Microsomal Glutathione Transferase 1. Spahiu L, Ålander J, Svensson R, Lehmer C, Armstrong R, Morgenstern R. [Manuscript]
History
Defence date
2014-06-09Department
- Institute of Environmental Medicine
Publisher/Institution
Karolinska InstitutetMain supervisor
Morgenstern, RalfPublication year
2014Thesis type
- Doctoral thesis
ISBN
978-91-7549-586-6Number of supporting papers
4Language
- eng