Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Abstract

Background: 5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/principal findings: Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC(50) 0.13 µM, 95% CI 0.08-0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC(50) 3.5 µM, 95% CI 2.3-5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC(50) values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions: CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.

Figure 1. Molecular structures of CAPE 1 and zileuton.

Figure 2. Summary of the synthesis of CAPE and its analogues.

Figure 3. Biosynthesis of 5-LO products by thapsigargin-stimulated PMNL in the presence of various compounds.

PMNL incubated with 1 µM of the indicated compounds or their diluent (Control, 0.5% DMSO) for 5 min were then stimulated with thapsigargin (1 µM) for 15 min in the presence (A) or absence (B) of exogenous arachidonic acid (10 µM). Dose-response of CAPE 1, compound 9 and zileuton for the inhibition of the biosynthesis of 5-LO products (C). Reactions were stopped by the addition of 0.5 volume of cold MeOH∶CH₃CN (1∶1) and samples were processed for measurement of 5-LO products by RP-HPLC. Total 5-LO products represent the sum of LTB₄, its trans isomers, 20-COOH- and 20-OH-LTB₄ and 5-hydroxyeicosatetraenoic acid. *Different from control, P<0.05, #different from control, P<0.005. AA = cells incubated without thapsigargin stimulus. Data are expressed as means ± SEM of 3 to 5 independent experiments, each performed in duplicate.

Figure 4. Impact of CAPE 1, compound 9 and zileuton on the synthesis of 5-LO products in cell lysates.

HEK293 cell lysate supernatants were incubated with CAPE 1, compound 9, zileuton or their diluent (Control, 0.5% DMSO). Synthesis of 5-LO products was initiated by the addition of 40 µM AA and 1 mM ATP. Reactions were stopped after 20 min by the addition of 0.5 volume of cold MeOH∶CH₃CN (1∶1) and samples were processed for measurement of 5-LO products by RP-HPLC. Total 5-LO products represent the sum of LTB₄, its trans isomers, 20-COOH- and 20-OH-LTB₄, and 5-hydroxyeicosatetraenoic acid. Values represent means ± SEM of three independent experiments, each performed in duplicate.

Figure 5. Impact of CAPE 1, compound 9 and zileuton on AA release by stimulated PMNL.

PMNL were incubated with 1 µM of the indicated compounds or their diluent (Control, 0.5% DMSO) for 5 min and were then stimulated with thapsigargin (1 µM) or its diluent (-thaps) for 5 min. Stimulation was stopped by the addition of 2 volumes of cold methanol containing octadeuterated-AA as an internal standard. Samples were stored at -20°C overnight, AA was extracted on octadecyl columns, pentafluorobenzylesters were prepared and were measured by GC-MS. *Different from control, P<0.05. Data are expressed as means ± SEM of three independent experiments, each performed in duplicate.

Figure 6. Impact of CAPE 1, compound 9 and zileuton on the biosynthesis of 5-LO products in stimulated whole blood.

Whole blood incubated with 1 µM of the indicated compounds or their diluent (Control, 0.5% DMSO) for 5 min was then stimulated with opsonised zymosan (5 mg/ml) for 30 min (A). Dose-response for the inhibition of 5-LO products of test compounds in opsonised zymosan-stimulated whole blood (B). After stimulation, blood was centrifuged, plasma was removed and added to 3.5 volumes of cold MeOH∶CH₃CN (1∶1) and samples were processed for measurement of 5-LO products by RP-HPLC. Total 5-LO products represent the sum of LTB₄, its trans isomers, 20-COOH- and 20-OH-LTB₄ and 5-hydroxyeicosatetraenoic acid. *Significantly different from control, P<0.05, #Significant different from control P<0.005. Data are expressed as means ± SEM of 3 independent experiments, each performed in duplicate.

Figure 7. Free radical scavenging and antioxidant activities of various test compounds.

(A) For free radical scavenging activity, 1 ml of DPPH (60 mM in ethanol) was mixed with 1 ml of the test compounds or their diluent (DMSO) in ethanol. Solutions were held in the dark for 30 min at room temperature and the absorbance was then measured at 520 nm. The free radical scavenging activity was expressed in terms of % inhibition of DPPH absorbance. (B) For antioxidant activity, test compounds or their diluent (DMSO) were added to a solution containing 0.16 mM linoleic acid and the oxidation reaction was initiated by adding 50 µl AAPH solution (10 mg/ml) to 1 ml of the above solution. The rate of lipid oxidation was determined by measuring the increase in absorbance at 234 nm over a 3 h period. Values represent the mean ± SEM of 3 independent experiments, each performed in triplicate.