In our ongoing effort to develop more potent inhibitors of leukotriene biosynthesis, CAPE 1
and several of its analogues were synthesized and compared to the reference molecule zileuton. The present study focused on the inhibition of 5-LO and LT biosynthesis in human PMNL as these cells are important producers of the powerful chemoattractant LTB4
. CAPE 1
was a more potent inhibitor than zileuton of 5-LO activity and of LT biosynthesis in stimulated human PMNL, although its inhibition of LT biosynthesis in whole blood was similar to that of zileuton. CAPE 1
had been previously reported to inhibit a plant lipoxygenase using linoleic acid as a substrate 
. Unfortunately, the authors reported this activity to be that of 5-LO. This was not the case since linoleic acid with its 9, 12 cis
double bond is not a 5-LO substrate and the product of the plant lipoxygenase-catalyzed reaction, 9-hydroperoxy-10, 12-octadecadienoic acid, is not a 5-LO product. 5-LO catalyzes the abstraction of a pro-S hydrogen at the C-7 position of substrates with 5, 8 cis
double bonds, like arachidonic acid, followed by the addition of molecular oxygen to form a 5-hydroperoxy-fatty acid 
. Therefore, the present study is the first report of the inhibition of 5-LO activity by CAPE.
Several structural features of CAPE 1
were investigated for their importance in the inhibition of LT biosynthesis and of 5-LO activity. The phenethyl ester group of the molecule was essential for effective inhibition since caffeic acid and cinnamic acid did not show significant activity at concentrations up to 10 µM (data not shown), and as previously reported in 5-LO-transfected HEK293 cells 
. This result in human leukocytes is not consistent with that reported in ionophore-stimulated murine peritoneal macrophages where both caffeic acid and CAPE show similar inhibition of leukotriene synthesis 
; the reason for this difference is not apparent but the phenethyl moiety is clearly required for the inhibition of both 5-LO activity and LT biosynthesis in human cells. Similarly, the catechol moiety of the molecule appears to be essential for activity as compounds 3
were without inhibitory activity at concentrations up to 10 µM (not shown). While compound 9
inhibited LT biosynthesis and 5-LO activity, the presence of the amide linkage reduced its potency compared to the ester CAPE 1
by approximately 3-fold for LT biosynthesis and by 18-fold for the inhibition of 5-LO activity in cell lysates ().
Human PMNL were stimulated in the presence or in the absence of exogenous AA to evaluate the inhibition of LT biosynthesis while bypassing the critical step of AA release from phospholipids, or not. CAPE 1
, zileuton and compound 9
were all more effective inhibitors of LT biosynthesis in the absence of exogenous AA suggesting that all three compounds inhibit the 5-LO catalyzed conversion of AA to LTs. A cell-free 5-LO assay using HEK293 cells that were stably transfected with 5-LO confirmed that CAPE 1
, zileuton and compound 9
all inhibited the 5-LO-catalyzed conversion of AA to LTs, where CAPE 1
0.13 µM) was 27-fold more active than zileuton for the inhibition of 5-LO activity while compound 9
and zileuton showed similar inhibitory activities.
It is well documented that human PMNL spontaneously release significant amounts of adenosine when they are cultured in vitro 
. This build up is usually not observed in tissues and blood since stromal cells and erythrocytes rapidly transport adenosine into their cytosol 
. This adenosine acts through G-protein linked receptors to activate adenylate cyclase and increase cellular cAMP levels 
. In human PMNL, elevated cAMP reduces numerous functional responses to agonist stimulation including oxygen radical (superoxide) production, phagocytosis and leukotriene biosynthesis 
. Therefore, freshly isolated human PMNL will quite rapidly (within minutes) begin to lose their capacity to respond to agonists unless the accumulation of adenosine in cell culture is prevented. We routinely add ADA to isolated PMNL incubations to prevent the inhibitory constraint of adenosine 
and although the possibility exists that added ADA may interact with and impact on the test compounds in the present study, this is unlikely since the inhibition of 5-LO activity and LT biosynthesis were also measured in broken cell preparations and in stimulated whole blood, two assay conditions that were devoid of added ADA.
Since the 5-LO inhibitor zileuton was previously shown to also inhibit AA release from membrane phospholipids in mouse peritoneal macrophages 
, the release of free AA from PMNL phospholipids was also measured following cell stimulation. CAPE 1
(1 µM) inhibited almost 55% of the AA released from membrane phospholipids of stimulated human PMN compared to 35% of inhibition with zileuton and no effect of compound 9
. Since the group IVA phospholipase A2
α) is responsible for AA release in stimulated human PMNL 
, these results suggest that both CAPE 1
and zileuton block LT biosynthesis in human PMN by inhibiting the activation of cPLA2
α as well as that of 5-LO.
Many 5-LO inhibitors including zileuton 
inhibit the enzyme by reducing the catalytically-active ferric form of 5-LO. Catechols as a class of compounds are known anti-oxidants and can potentially inhibit 5-LO as free radical scavengers and antioxidants. When the test compounds were evaluated for anti-oxidant and free-radical scavenging activity, their potency was not necessarily related to their ability to inhibit 5-LO. Not surprisingly, cinnamic acid and its phenethyl ester and phenethyl amide derivatives, compounds 3
, showed no antioxidant or free radical scavenging activity at concentrations up to 25 µM and 100 µM, respectively. However, caffeic acid, which showed no inhibition of 5-LO, was as effective an antioxidant and free-radical scavenger as CAPE 1
and compound 9
. This suggests that catechols may not efficiently reduce the ferric iron of 5-LO for enzyme inhibition without the contribution of a hydrophobic moiety that may act as an anchor to more specifically target the non-heme iron of the 5-LO protein. Zileuton was also an effective anti-oxidant as previously reported, but was not a good free radical scavenger.
The rationale for synthesizing amide-linked analogues was that they may be more stable than ester-linked compounds, like CAPE 1, which could be susceptible to hydrolysis by esterases. Despite being much more potent than zileuton and compound 9 at inhibiting 5-LO activity in broken cell assays, and despite inhibiting the release of AA from stimulated cells, CAPE 1 was only moderately more effective than compound 9 at inhibiting LT biosynthesis in stimulated human PMNL and in whole blood, and was not different from zileuton in whole the blood assays. This suggests that while remaining a potent inhibitor of LT biosynthesis, the suspected susceptibility of CAPE 1 to esterases may reduce its potency in a physiological setting.
In summary, we characterized CAPE 1
, a naturally occurring component of propolis from honeybee hives, as a potent inhibitor of LT biosynthesis that acts as a dual inhibitor of 5-LO activity and of AA release from membrane phospholipids. A continued effort for the rational design of inhibitors of LT biosynthesis using the CAPE 1
structure as framework may yield stable and potent inhibitors of LT biosynthesis. Such rational design efforts will certainly be aided by the recent description of the crystal structure of the human 5-LO protein