Modifications in FLAP 's second cytosolic loop influence 5‐ LOX interaction, inhibitor binding, and leukotriene formation

Leukotrienes, synthesized via the 5‐lipoxygenase (5‐LOX) pathway in the arachidonic acid cascade, are critical in inflammation. Effective leukotriene production requires interaction between 5‐LOX and 5‐LOX‐activating protein (FLAP) at the nuclear membrane. This study used site‐directed mutagenesis to explore amino acid residues in FLAP's inhibitor binding pocket and cytosolic loops, assessing their impact on 5‐LOX product formation, the FLAP inhibitor MK886's efficacy, 5‐LOX translocation, and 5‐LOX/FLAP complex formation. Mutations in the second cytosolic loop, especially at residue S108, reduced MK886 potency and disrupted 5‐LOX/FLAP complex formation. These results highlight the second cytosolic loop of FLAP in the 5‐LOX/FLAP interaction and proper leukotriene formation and suggest that targeting this region could aid in the development of new FLAP inhibitors with improved pharmacokinetics.

The enzyme 5‐lipoxygenase (5‐LOX) catalyzes the first step in the biosynthesis of leukotrienes (LTs) involved in inflammatory pathophysiology. After cellular stimulation, 5‐LOX translocates to the nucleus, interacting with the 5‐LOX‐activating protein (FLAP) to form LTA 4 from arachidonic acid (AA). Here, we found that the C2‐loop, and in particular position S108 of FLAP, is crucial for the interaction with 5‐LOX.