PHARMACOLOGICAL CROSS-REACTIVITY BETWEEN 5-LIPOXYGENASE-ACTIVATING PROTEIN, 5-LIPOXYGENASE, AND LEUKOTRIENE C-4 SYNTHASE

Leukotriene (LT) C-4 synthase, an integral membrane protein, catalyzes the first committed step in the biosynthesis of the peptidyl leukotri enes, which have been implicated in various inflammatory disorders, in cluding human bronchial asthma. To identify possible inhibitors of LTC 4 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 assay s 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 rever se-phase high pressure liquid chromatography. Representative compounds from distinct structural classes were tested over a concentration ran ge of 40 nM to 100 mu M. The most potent inhibitor was found to be a p reviously established nanomolar 5-lipoxygenase inhibitor, 1H-thiopyran o[2,3,4-c,d]indol-2-yl]ethoxy]butanoic acid (L-699,333) of the phenylp yridine structural class of compounds. L-699,333 inhibited LTC4 syntha se 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 thi s enzyme. Structure-activity analyses of other phenylpyridines indicat ed that the inhibition imparted by L-699,333 was retained following th e replacement of the carboxylic acid group with other equivalents. Str ucturally diverse FLAP inhibitors tested against LTC4 synthase were al l micromolar inhibitors of the enzyme over a 10-fold range, with MK-88 6 at 11 mu M. These results implicate that compounds that bind competi tively to arachidonic acid binding sites on FLAP and 5-LO recognize mo tifs that are also weakly conserved on the binding site of LTC4 syntha se.