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Leukotriene C 4 synthase (LTC4S) catalyzes the formation of the proinflammatory lipid mediator leukotriene C 4 (LTC 4 ). LTC 4 is the parent molecule of the cysteinyl leukotrienes, which are recognized for their pathogenic role in asthma and allergic diseases. Cellular LTC4S activity is suppressed by PKC-mediated phosphorylation, and recently a downstream p70S6k was shown to play an important role in this process. Here, we identified Ser 36 as the major p70S6k phosphorylation site, along with a low frequency site at Thr 40 , using an in vitro phosphorylation assay combined with mass spectrometry. The functional consequences of p70S6k phosphorylation were tested with the phosphomimetic mutant S36E, which displayed only about 20% (20 mol/min/mg) of the activity of WT enzyme (95 mol/min/mg), whereas the enzyme activity of T40E was not significantly affected. The enzyme activity of S36E increased linearly with increasing LTA 4 concentrations during the steady-state kinetics analysis, indicating poor lipid substrate binding. The Ser 36 is located in a loop region close to the entrance of the proposed substrate binding pocket. Comparative molecular dynamics indicated that Ser 36 upon phosphorylation will pull the first luminal loop of LTC4S toward the neighboring subunit of the functional homotrimer, thereby forming hydrogen bonds with Arg 104 in the adjacent subunit. Because Arg 104 is a key catalytic residue responsible for stabilization of the glutathione thiolate anion, this phosphorylation-induced interaction leads to a reduction of the catalytic activity. In addition, the positional shift of the loop and its interaction with the neighboring subunit affect active site access. Thus, our mutational and kinetic data, together with molecular simulations, suggest that phosphorylation of Ser 36 inhibits the catalytic function of LTC4S by interference with the catalytic machinery. Leukotriene (LT)2 C 4 synthase (LTC4S) catalyzes the formation of LTC 4 by conjugating the unstable allylic epoxide intermediate LTA 4 with reduced glutathione (GSH) (1). LTC 4 and its metabolites LTD 4 and LTE 4 are known as cysteinyl leukotrienes (cys-LTs), which are involved in bronchial asthma and allergic inflammatory disorders (1-3). The cys-LTs signal through two G-protein-coupled receptors, denoted CysLT1 and CysLT2, to exert their biological functions such as smooth muscle contraction and increased vascular permeability. Several drugs, typified by montelukast, have been developed that specifically target the CysLT1 receptor (4). Recently, additional G-protein-coupled receptors that recognize cys-LTs have been identified, in particular gpr17 and CysLT3 (5, 6). The increasing complexity of cys-LT signaling has promoted research and drug development efforts targeting the upstream LTC4S as it catalyzes the committed step in cys-LT biosynthesis (7).The leukotrienes are derived from arachidonic acid through the 5-lipoxygenase pathway where cytosolic phospholipase A 2 , 5-lipoxygenase, and 5-lipoxygenase-activating protein play important rol...

Cysteinyl leukotrienes (cys-LTs) are lipid mediators of inflammation. The enzyme catalyzing synthesis of cys-LTs, leukotriene C 4 synthase (LTC4S), is considered an important drug target. Here we report the synthesis and characterization of three tandem benzophenone amino pyridines as inhibitors of LTC4S in vitro and in vivo. The inhibitors were characterized in vitro using recombinant human LTC4S, MonoMac 6 cells, and a panel of peripheral human immune cells. In vivo, the compounds were tested in the Zymosan A-induced peritonitis mouse model. The molecules, denoted TK04, TK04a, and TK05, were potent and selective inhibitors of LTC4S with IC 50 values of 116, 124, and 95 nM, respectively. Molecular docking revealed binding in a hydrophobic crevice between two enzyme monomers and interaction with two catalytic residues, Arg104 and Arg31. The TK compounds potently inhibited cys-LT biosynthesis in immune cells. In coincubations of platelets and polymorphonuclear leukocytes, inhibition of LTC4S led to shunting of LTA 4 toward anti-inflammatory lipoxin A 4 , which was significantly enhanced by simultaneous inhibition of LTA4H. Finally, we found that TK05 (6 mg×kg 21 ×body weight) reduces LTE 4 levels in peritoneal lavage fluid by 88% and significantly decreases vascular permeability in vivo. Our findings indicate that the TK compounds are valuable experimental tools in eicosanoid research in vitro and in vivo. Their chemical structures may serve as leads for further inhibitor design. Novel drugs depleting cys-LT production could be beneficial for treatment of inflammatory diseases associated with overexpression of LTC4S.

Microsomal glutathione S-transferase 2 (MGST2) produces leukotriene C4, key for intracrine signaling of endoplasmic reticulum (ER) stress, oxidative DNA damage and cell death. MGST2 trimer restricts catalysis to only one out of three active sites at a time, but the molecular basis is unknown. Here, we present crystal structures of human MGST2 combined with biochemical and computational evidence for a concerted mechanism, involving local unfolding coupled to global conformational changes that regulate catalysis. Furthermore, synchronized changes in the biconical central pore modulate the hydrophobicity and control solvent influx to optimize reaction conditions at the active site. These unique mechanistic insights pertain to other, structurally related, drug targets.

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100.