Tandem Benzophenone Amino Pyridines, Potent and Selective Inhibitors of Human Leukotriene C₄ Synthase

Abstract

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.

Current and future biomarkers in allergic asthma

“…Therefore, eosinophil-based diagnosis benefits from additional assessment of surface markers such as CD69 (50) and sputum mediator profiles (e.g. cysteinyl leukotrienes, prostaglandin D 2 , IL-13), which facilitate the differentiation between asthma phenotypes or endotypes and predicted responsiveness to treatment with current leukotriene modifiers such as montelukast or potential future therapeutics, for example enzyme inhibitors or certain monoclonal anticytokine antibodies (51)(52)(53). As leukotrienes are recognized as central mediators in neonatal and childhood phenotypes, the measurement of sputum leukotriene levels may be particularly promising to monitor the development of asthmatic inflammation early in life (54,55).…”

Discovery of the Oral Leukotriene C4 Synthase Inhibitor (1S,2S)-2-({5-[(5-Chloro-2,4-difluorophenyl)(2-fluoro-2-methylpropyl)amino]-3-methoxypyrazin-2-yl}carbonyl)cyclopropanecarboxylic Acid (AZD9898) as a New Treatment for Asthma

“…Boosting of LXA4 levels have been previously reported in an unstimulated isolated platelet/neutrophil cell mixture treated with an LTC4S inhibitor TA05 . However, the increase in lipoxins levels on TA05 treatment did not show a consistent and statistically significant dose-related response and only achieved a significant change with the addition of an LTA4H inhibitor.…”

Phosphorylation of Leukotriene C4 Synthase at Serine 36 Impairs Catalytic Activity

“…The observed behavior with the S36E mutant indicates that phosphorylation at Ser 36 may interfere with TK04 binding, thus effecting the inhibitor potency. TK04 was proposed to occupy the LTA 4 binding site and interact with Arg 104 based on molecular docking results (30). The efficiency of the TK04 inhibitor at concentrations around or below IC 50 seems reduced with the phosphomimetic S36E mutant, which is yet an indication that phosphorylation affects lipid substrate binding.…”