Pharmacological cross-reactivity between 5-lipoxygenase-activating protein, 5-lipoxygenase, and leukotriene C4 synthase

Leukotriene (LT) C-4 synthase, an integral membrane protein, catalyzes the first committed step in the biosynthesis of the peptidyl leukotrienes, which have been implicated in various inflammatory disorders, including human bronchial asthma. To identify possible inhibitors of LTC4 synthase, synthetic compounds known to inhibit other proteins in the leukotriene biosynthetic pathway (5-lipoxygenase-activating protein, FLAP, and 5-lipoxygenase, 5-LO) or to antagonize leukotriene receptors (cys LT1) were tested for activity against LTC4 synthase. These assays were performed on enriched fractions of human LTC4 synthase purified from the human monocytic cell line THP-1. LTA(4) and glutathione were used as substrates, and LTC4 product formation was monitored by reverse-phase high pressure liquid chromatography. Representative compounds from distinct structural classes were tested over a concentration range of 40 nM to 100 mu M. The most potent inhibitor was found to be a previously established nanomolar 5-lipoxygenase inhibitor, 2-[2-[1-(4-chlorobenzyl)-4-methyl-6-[(5-phenylpyridin-2-yl)-methoxy]-4,5-dihydro-1H-thiopyrano[2,3,4-c,d]indol-2-yl]ethoxy]butanoic acid (L-699,333) of the phenylpyridine structural class of compounds. L-699,333 inhibited LTC4 synthase activity in vitro with an IC50 value of 3.9 mu M and a K-i value of 0.25 mu M, making it the most potent synthetic inhibitor known of this enzyme. Structure-activity analyses of other phenylpyridines indicated that the inhibition imparted by L-699,333 was retained following the replacement of the carboxylic acid group with other equivalents. Structurally diverse FLAP inhibitors tested against LTC4 synthase were all micromolar inhibitors of the enzyme over a 10-fold range, with MK-886 at 11 mu M. These results implicate that compounds that bind competitively to arachidonic acid binding sites on FLAP and 5-LO recognize motifs that are also weakly conserved on the binding site of LTC4 synthase.