Identification of Peracetylated Quercetin as a Selective 12-Lipoxygenase Pathway Inhibitor in Human Platelets.

The inflammatory response is necessary for the host's defense against pathogens; however, uncontrolled or unregulated production of eicosanoids has been associated with several types of chronic inflammatory diseases. Thus, it is not surprising that enzymes implicated in the production of eicosanoids have been strategically targeted for potential therapeutic approaches. The 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] lipid mediator is among inflammatory molecules that are abundantly produced in various diseases and is primarily biosynthesized via the 12(S)-lipoxygenase pathway. The effects of the abundance of 12(S)-HETE and its contribution to several chronic inflammatory diseases have been well studied over the last few years. While most developed compounds primarily target the 5-lipoxygenase (5-LO) or the cyclooxygenase (COX) pathways, very few compounds selectively inhibiting the 12-lipoxygenase (12-LO) pathway are known. In this study, we examined whether the distribution of hydroxyl groups among flavones could influence their potency as 12-LO inhibitors. Using human platelets, the human embryonic kidney 293 (HEK293) cell line expressing 5-LO, and human polymorphonuclear leukocytes (PMNLs) we investigated the effects of these compounds on several inflammatory pathways, namely, 12-LO, 5-LO, and COX. Using high-resolution respirometry and flow cytometry, we also evaluated some normal cell functions that could be modulated by our compounds. We identified a peracetylated quercetin (compound 6) that exerts potent inhibitory activity toward the platelet 12-LO pathway (IC50 = 1.53 µM) while having a lesser affinity toward the COX pathway. This study characterizes the peracetylated quercetin (compound 6) as a more selective platelet-type 12-LO inhibitor than baicalein, with no measurable nontargeted effects on the platelet's activation or overall cell's oxygen consumption.

On the cellular metabolism of the click chemistry probe 19-alkyne arachidonic acid.

Alkyne and azide analogs of natural compounds that can be coupled to sensitive tags by click chemistry are powerful tools to study biological processes. Arachidonic acid (AA) is a FA precursor to biologically active compounds. 19-Alkyne-AA (AA-alk) is a sensitive clickable AA analog; however, its use as a surrogate to study AA metabolism requires further evaluation. In this study, AA-alk metabolism was compared with that of AA in human cells. Jurkat cell uptake of AA was 2-fold greater than that of AA-alk, but significantly more AA-Alk was elongated to 22:4. AA and AA-alk incorporation into and remodeling between phospholipid (PL) classes was identical indicating equivalent CoA-independent AA-PL remodeling. Platelets stimulated in the pre-sence of AA-alk synthesized significantly less 12-lipoxygenase (12-LOX) and cyclooxygenase products than in the presence of AA. Ionophore-stimulated neutrophils produced significantly more 5-LOX products in the presence of AA-alk than AA. Neutrophils stimulated with only exogenous AA-alk produced significantly less 5-LOX products compared with AA, and leukotriene B4 (LTB4)-alk was 12-fold less potent at stimulating neutrophil migration than LTB4, collectively indicative of weaker leukotriene B4 receptor 1 agonist activity of LTB4-alk. Overall, these results suggest that the use of AA-alk as a surrogate for the study of AA metabolism should be carried out with caution.

LPS induces ALOX5 promoter activation and 5-lipoxygenase expression in human monocytic cells.

5-lipoxygenase (5-LO), coded by the ALOX5 gene, is expressed in leukocytes and catalyzes the formation of leukotrienes, pro-inflammatory lipid mediators. Leukotrienes are central to immune responses, but are also involved in inflammatory disorders and 5-LO expression is associated with leukemia stem cell survival. It is therefore important to understand mechanisms that control 5-LO expression. This study investigated the control of 5-LO expression and leukotriene biosynthesis following the maturation of human monocytic cells. MonoMac-1 (MM1) and THP-1 cells were incubated for up to 72 h with or without LPS and TGF-ß. LPS, but not TGF-ß, increased CD14 expression in both MM1 and THP-1 cells. Incubation with LPS (100 ng/ml) and TGF-ß (1 ng/ml) synergistically increased the capacity of MM1 cells to produce 5-LO products from undetectable levels to 40±5 pmol/106 cells. 5-LO product biosynthesis in THP-1 cells increased 25-fold. A synergistic effect of LPS and TGF-ß was measured with increases in 5-LO mRNA of 54- and 13-fold in MM1 and THP-1 cells, respectively. 5-LO protein expression increased significantly in both MM1 and THP-1 cells. ALOX5 promoter activity was significantly elevated >2-fold in both cell lines following LPS treatment, but TGF-ß was without effect. The main 5-LO products were cysteinyl-leukotrienes, however LPS and TGF-ß did not impact on the capacity of the cells to metabolize leukotriene A4. Overall, this study demonstrates that receptor-mediated stimulation of MM1 and THP-1 cells by LPS is associated with increased 5-LO expression. This represents a new mechanism by which leukotriene biosynthesis can be modulated by pathological agents.

Phenolic acid phenethylesters and their corresponding ketones: Inhibition of 5-lipoxygenase and stability in human blood and HepaRG cells.

5-lipoxygenase (5-LO) catalyzes the biosynthesis of leukotrienes, potent lipid mediators involved in inflammatory diseases, and both 5-LO and the leukotrienes are validated therapeutic targets. Caffeic acid phenethyl ester (CAPE) is an effective inhibitor of 5-LO and leukotriene biosynthesis but is susceptible to hydrolysis by esterases. In this study a number of CAPE analogues were synthesized with modifications to the caffeoyl moiety and the replacement of the ester linkage with a ketone. Several new molecules showed better inhibition of leukotriene biosynthesis than CAPE in isolated human neutrophils and in whole blood with IC50 values in the nanomolar (290-520 nmol/L) and low micromolar (1.0-2.3 µmol/L) ranges, respectively. Sinapic acid and 2,5-dihydroxy derivatives were more stable than CAPE in whole blood, and ketone analogues were degraded more slowly in HepaRG hepatocyte cultures than esters. All compounds underwent modification consistent with glucuronidation in HepaRG cultures as determined using LC-MS/MS analysis, though the modified sinapoyl ketone (10) retained 50% of its inhibitory activity after up to one hour of incubation. This study has identified at least one CAPE analogue, compound 10, that shows favorable properties that warrant further in vivo investigation as an antiinflammatory compound.

Clicked cinnamic/caffeic esters and amides as radical scavengers and 5-lipoxygenase inhibitors.

5-Lipoxygenase (5-LO) is the key enzyme responsible for the conversion of arachidonic acid to leukotrienes, a class of lipid mediators implicated in inflammatory disorders. In this paper, we describe the design, synthesis, and preliminary activity studies of novel clicked caffeic esters and amides as radical scavengers and 5-LO inhibitors. From known 5-LO inhibitor 3 as a lead, cinnamic esters 8a-h and amides 9a-h as well as caffeic esters 15a-h and amides 16a-h were synthesized by Cu(I)-catalyzed [1,3]-dipolar cycloaddition with the appropriate azide precursors and terminal alkynes. All caffeic analogs are proved to be good radical scavengers (IC50: 10-20 µM). Esters 15g and 15f possessed excellent 5-LO inhibition activity in HEK293 cells and were equipotent with the known 5-LO inhibitor CAPE and more potent than Zileuton. Several synthesized esters possess activities rivaling Zileuton in stimulated human polymorphonuclear leukocytes.