title: Development of smart cell-free and cell-based assay systems for investigation of leukotriene C4synthase activity and evaluation of inhibitors.
abstract: Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A4(LTA4) that is subsequently conjugated with glutathione (GSH) by LTC4synthase (LTC4S). Cys-LT receptor antagonists and LTC4S inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA4have hampered the development of LTC4S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA4that allow studying LTC4S activity and investigating LTC4S inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTC4S with isolated 5-LOX efficiently converted exogenous AA to LTC4(~1.3μg/200μg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC4S with Ca2+-ionophore A23187 and 20μM AA resulted in strong LTC4formation (~250ng/106cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC4S, consistently inhibited LTC4formation in all assay types (IC50=3.1-3.5μM) and we successfully confirmed TK04a as potent LTC4S inhibitor in these assay systems (IC50=17 and 300nM, respectively). We demonstrated transcellular LTC4biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA4from AA and HEK293 cells expressing LTC4S that transform LTA4to LTC4. In conclusion, our assay approaches are advantageous as the substrate LTA4is generated in situ and are suitable for studying enzymatic functionality of LTC4S including site-directed mutations and evaluation of LTC4S inhibitors.
authors.affiliation: Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Liening
authors.affiliation: Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany.
authors.forename: Gerhard K
authors.initials: GK
authors.lastname: Scriba
authors.affiliation: Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Rummler
authors.affiliation: Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Weinigel
authors.affiliation: Division of Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Thea K
authors.initials: TK
authors.lastname: Kleinschmidt
authors.affiliation: Division of Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggström
authors.affiliation: Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Werz
authors.affiliation: Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany. Electronic address: ulrike.garscha@uni-jena.de.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Garscha
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D017997:Leukotriene C4 / Q000378:metabolism; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D004791:Enzyme Inhibitors; D017997:Leukotriene C4; D001094:Arachidonate 5-Lipoxygenase; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase
keywords: Assay; HEK293 cells; Inhibitor; LTC(4) synthase; Leukotriene C(4)