Development of smart cell-free and cell-based assay systems for investigation of leukotriene C-4 synthase activity and evaluation of inhibitors

作者：Liening Stefanie; Scriba Gerhard K; Rummler Silke; Weinigel Christina; Kleinschmidt Thea K; Haeggstrom Jesper Z; Werz Oliver; Garscha Ulrike^*

来源：Biochimica et Biophysica Acta-Molecular and Cell Biology of Lipids, 2016, 1861(11): 1605-1613.

DOI：10.1016/j.bbalip.2016.07.011

• Leukotriene C-4
• LTC4 synthase
• Inhibitor
• Assay
• HEK293 cells

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 A(4) (LTA(4)) that is subsequently conjugated with glutathione (GSH) by LTC4 synthase (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 LTA(4) have hampered the development of LTC4S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA(4) that 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 (similar to 1.3 mu g/200 mu g protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC4S with Ca2+-ionophore A23187 and 20 mu M AA resulted in strong LTC4 formation (similar to 250 ng/10(6) cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC4S, consistently inhibited LTC4 formation in all assay types (IC50 = 3.1-3.5 mu M) and we successfully confirmed TK04a as potent LTC4S inhibitor in these assay systems (IC50 =17 and 300 nM, respectively). We demonstrated transcellular LTC4 biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA(4) from AA and HEK293 cells expressing LTC4S that transform LTA(4) to LTC4. In conclusion, our assay approaches are advantageous as the substrate LTA(4) is generated in situ and are suitable for studying enzymatic functionality of LTC4S including site-directed mutations and evaluation of LTC4S inhibitors.

• 出版日期2016-11