Effect of BI-L-239, A-64077 and MK-886 on leukotriene B4 synthesis by chopped guinea pig lung and on antigen-induced tracheal contraction in vitro.

The 5-lipoxygenase (5-LO) inhibitors BI-L-239 and A-64077 were compared with the 5-LO translocation inhibitor MK-886 for the ability to inhibit leukotriene B4 (LTB4) biosynthesis by chopped (1 mm3) guinea pig lung. LTB4 synthesis by ovalbumin-sensitized chopped lung tissue was determined after stimulation with either calcium ionophore (A23187) or antigen. With A23187 stimulation, MK-886 was more potent (IC50 = 0.39 +/- 0.23 microM, mean +/- SEM, p < 0.01) than BI-L-239 (IC50 = 2.48 +/- 0.46 microM) or A-64077 (IC50 = 4.68 +/- 0.70 microM) and BI-L-239 was more potent than A64077 (p < 0.02). Thus, the order of potency was MK-886 > BI-L-239 > A-64077 for inhibition of calcium ionophore-induced LTB4 generation. There was no significant differences in potency of the compounds in chopped lung stimulated with antigen: IC50 for LTB4 synthesis by A-64077 = 3.31 +/- 1.70 microM, for BI-L-239 = 9.06 +/- 4.94 microM, and for MK-886 = 13.33 +/- 7.91 microM. The ability of these compounds to inhibit contraction of tracheal tissue from actively sensitized guinea pigs in response to antigen was also determined in the presence of indomethacin (15 micrograms/ml), mepyramine, and atropine (5 micrograms each/ml). Both 5-LO inhibitors inhibited antigen-induced contraction, with IC50 values for BI-L-239 and A-64077 of 1.58 and 4.35 microM respectively. MK-886 was ineffective at inhibiting antigen-induced tracheal contraction in vitro at concentrations up to 30 microM. In summary, these compounds inhibit antigen-induced and A23187-induced leukotriene biosynthesis in guinea pig tissue. These 5-LO inhibitors were similarly effective at inhibiting antigen-induced tracheal contraction where MK-886 was ineffective.     

Design and synthesis of eugenol derivatives, as potent 15-lipoxygenase inhibitors

A group of 4-allyl-2-methoxyphenol (eugenol) esters were designed, synthesized, and evaluated as potential inhibitors of soybean 15-lipoxygenase (SLO). Compounds 4c, 4d 4f, 4p, and 4q showed the best IC(50) in SLO inhibition (IC(50)=1.7, 2.3, 2.1, 2.2, and 0.017microM, respectively). All compounds were docked into SLO active site and showed that allyl group of compounds is oriented toward the iron atom in the active site of SLO. It is assumed that lipophilic interaction of ligand-enzyme would be in charge of inhibiting the enzyme activity. The selectivity of eugenol derivatives in inhibiting 15-HLOb was also compared with 15-HLOa by molecular modeling and multiple alignment techniques.     

Indolizine 1-sulfonates as potent inhibitors of 15-lipoxygenase from soybeans

A number of indolizine 1-sulfonates have been prepared by cyclization of cyclopropenones with pyridines followed by trapping of the intermediate 1-indolizinol with a sulfonyl halide, and examined as inhibitors of 15-lipoxygenase (15-LO). The compounds display IC(50) values between 15 and 42 microM; all are more active than the well-known 15-LO inhibitor quercetin (IC(50) 51 microM). A wide variety of substituents are well tolerated. The enzyme inhibition was not affected by preincubation or the presence of a detergent and no significant particle formation was observed. Hence, inhibition from aggregates of indolizines, promiscuous inhibition, is highly unlikely.     

Identification of 2-mercaptohexanoic acids as dual inhibitors of 5-lipoxygenase and microsomal prostaglandin E₂ synthase-1

5-Lipoxygenase (5-LO) and microsomal prostaglandin E? synthase (mPGES)-1 are key enzymes in the biosynthesis of leukotrienes and prostaglandin (PG)E?, respectively, and are considered as valuable targets for the treatment of inflammatory diseases. Here, we present the identification of 2-mercaptohexanoic acid derivatives as dual inhibitors of 5-LO and mPGES-1. The lead compound 2(4-(3-biphenyloxypropoxy)phenylthio)hexanoic acid (21) inhibits human 5-LO and mPGES-1 in cell-free assays with an IC?? = 3.5 and 2.2 μM, respectively, and suppresses 5-LO in intact cells with even a higher potency (IC??=0.9 μM). Compound 21 (10 μM) neither significantly inhibited the related 12- or 15-LOs nor cyclooxygenase-1 and -2 or cytosolic phospholipase A?. Based on the selective and potent inhibition of 5-LO and mPGES-1, further assessment of these 2-mercaptohexanoic acids in preclinical models of inflammation are warranted.     

Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol- 2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes.

We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-i ndol-2- yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or formyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its omega-oxidation products, and 6-trans-isomers with IC50 values of 2.8-4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 microM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its omega-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5-11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3-0.9, 3.7-5.3, and 8.5-17.3 nM, respectively. In neutrophils primed with granulocyte--macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7-8.7 nM. At the concentration of 1 microM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.     

Synthesis and biological evaluation of a class of 5-benzylidene-2-phenyl-thiazolinones as potent 5-lipoxygenase inhibitors

A class of 5-lipoxygenase (5-LO) inhibitors characterized by a central 5-benzylidene-2-phenyl-thiazolinone scaffold was synthesized as a new series of molecular modifications and extensions of a previously reported series. Compounds were tested in a cell-based and a cell-free assay and furthermore evaluated for their influence on cell viability. The presented substituted thiazolinone scaffold turned out to be essential for both the 5-LO inhibitory activity and the non-cytotoxic profile. With (Z)-5-(4-methoxybenzylidene)-2-(naphthalen-2-yl)-5H-thiazol-4-one (2k, ST1237), a potent, direct, non-cytotoxic 5-LO inhibitor with IC(50) of 0.08 μM and 0.12 μM (cell-free assay and intact cells), we present a promising lead optimization and development for further investigations as novel anti-inflammatory drug.     

Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP)

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 μM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 μM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.     

SAR-study on a new class of imidazo[1,2-a]pyridine-based inhibitors of 5-lipoxygenase

A novel class of 5-lipoxygenase (5-LO) inhibitors characterized by a central imidazo[1,2-a]pyridine scaffold, a cyclohexyl moiety and an aromatic system, is presented. This scaffold was identified in a virtual screening study and exhibits promising inhibitory potential on the 5-LO. Here, we investigate the structure-activity relationships of this compound class. With N-cyclohexyl-6-methyl-2-(4-morpholinophenyl)imidazo[1,2-a]pyridine-3-amine (14), we identified a potent 5-LO inhibitor (IC(50)=0.16μM (intact cells) and 0.1μM (cell-free)), which may possess potential as an effective lead compound intervening with inflammatory diseases and certain types of cancer.     

Indolizines as novel potent inhibitors of 15-lipoxygenase

15-Lipoxygenase (15-LO) has been implicated in oxidation of low-density lipoproteins (LDL) and this enzyme may be involved in the development of atherosclerosis. We have examined 1-substituted indolizines as possible inhibitors of 15-LO from soy beans and from rabbit reticulocytes. Most compounds studied were significantly more active as inhibitors of 15-LO from soy beans than quercetin. The indolizines were slightly less potent inhibitors of the mammalian enzyme, but we found good correlation between inhibitory activity against both 15-LO enzymes studied. Several of the compounds were only weak DPPH scavengers and they may therefore be regarded as so-called non antioxidant inhibitors of 15-LO.     

New bis-catechols 5-lipoxygenase inhibitors

Three polyhydroxy-2-phenylnaphthalenes (1-3) and the oxy analogue of tetrahydroxypavinan (4) were prepared and evaluated for their antioxidant properties (inhibition of diphenylpycrylhydrazyl radical (DPPH), reduction of iron (III) ion) and inhibition of 5-lipoxygenase (5-LO) activity. Their three-dimensional structures were established on the basis of spectroscopic data and semiempirical calculations. Compounds 1 and 2 were found as potent 5-LO inhibitors as nordihydroguaiaretic acid (NDGA), whereas 4 is 2.5 times less potent than NDGA. The reliability of the 3-D structures with the 5-LO inhibition properties is discussed. Their antioxidant properties show that tested compounds are expected to act as redox inhibitors.     

Synthesis and biological evaluation of acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl pharmacophore: dual inhibitors of cyclooxygenases and lipoxygenases

A new class of regioisomeric acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl (DTBHP) 5-lipoxygenase (5-LOX) pharmacophore that is vicinal to a para-methanesulfonylphenyl cyclooxygenase-2 (COX-2) pharmacophore were designed for evaluation as selective COX-2 and/or 5-LOX inhibitors. The target compounds were synthesized via a highly stereoselective McMurry olefination cross-coupling reaction. This key synthetic step afforded a (Z):(E) olefinic mixture with a predominance for the (Z)-olefin stereoisomer. Structure-activity studies for the (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-methanesulfonylphenyl)-1-phenylalk-1-ene regioisomers showed that COX-1 inhibition decreased, COX-2 inhibition increased, and the COX-2 selectivity index (SI) increased as the chain length of the alkyl substituent attached to the olefinic double bond was increased (Et-->n-butyl-->n-heptyl). In this group of compounds, inhibition of both 5-LOX and 15-LOX was dependent upon the length of the alkyl substituent with the hex-1-ene compound 9c having a n-butyl substituent exhibiting potent inhibition of both 5-LOX (IC50=0.3 microM) and 15-LOX (IC50=0.8 microM) relative to the inactive (IC50>10 microM) Et and n-heptyl analogs. Compound 9c is of particular interest since it also exhibits a dual inhibitory activity against the COX (COX-1 IC50=3.0 microM, and COX-2 IC50=0.36 microM, COX-2 SI=8.3) isozymes. A comparison of the relative inhibitory activities of the two groups of regioisomers investigated shows that the regioisomers in which the alkyl substituent is attached to the same olefinic carbon atom (C-2) as the para-methanesulfonylphenyl moiety generally exhibit a greater potency with respect to COX-2 inhibition. The 4-hydroxy substituent in the 3,5-di-tert-butyl-4-hydroxyphenyl moiety is essential for COX and LOX inhibition since 3,5-di-tert-butyl-4-acetoxyphenyl derivatives were inactive inhibitors. These structure-activity data indicate acyclic triaryl (Z)-olefins constitute a suitable template for the design of dual COX-2/LOX inhibitors.     

Antioxidant activity of synthetic cytokinin analogues: 6-alkynyl- and 6-alkenylpurines as novel 15-Lipoxygenase inhibitors

Synthetic cytokinin analogues as well as the well known CKs 6-benzylaminopurine (BAP), kinetin and trans-zeatin were examined for antioxidant activity. The compounds were tested as potential diphenylpicrylhydrazyl (DPPH) scavengers and as inhibitors of 15-lipoxygenase (15-LO). The natural plant hormones were essentially inactive in both assays, but several synthetic analogues have a profound inhibiting effect on 15-lipoxygenase from soybeans. The same compounds were only weak DPPH scavengers and they may therefore be regarded as so-called non antioxidant inhibitors of 15-LO.     

Inhibition of lipoxygenases and cyclooxygenases by linoleyl hydroxamic acid: comparative in vitro studies

In this first comparative in vitro study, linoleyl hydroxamic acid (LHA), a simple and stable derivative of linoleic acid, was tested as an inhibitor of several enzymes involved in arachidonic acid metabolism in mammals. The tested enzymes were human recombinant 5-lipoxygenase (h5-LO), porcine leukocyte 12-LO, rabbit reticulocyte 15-LO, ovine cyclooxygenases 1/2 (COX1/COX2), and human microsomal prostaglandin E synthase-1 (mPGES-1). Potato tuber and soybean lipoxygenases (ptLOX and sLOX, respectively) were studied for comparative purposes. LHA inhibited most of the tested enzymes with the exception of mPGES-1. The LHA inhibitory activity increased as follows: mPGES-1 (no inhibition)相似文献   

Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416

U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.     

Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416

U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.     

Platelet-derived growth factor (PDGF) induces pulmonary vascular remodeling through 15-LO/15-HETE pathway under hypoxic condition

15-lipoxygenase (15-LO) is known to play an important role in chronic pulmonary hypertension. Accumulating evidence for its down-stream participants in the vasoconstriction and remodeling processes of pulmonary arteries, while how hypoxia regulates 15-LO/15-hydroxyeicosatetraenoic acid (15-HETE) to mediate hypoxic pulmonary hypertension is still unknown. Platelet-derived growth factor (PDGF) is an important vascular regulator whose concentration increases under hypoxic condition in the lungs of both humans and mice with pulmonary hypertension. The present study was carried out to determine whether hypoxia advances the pulmonary vascular remodeling through the PDGF/15-LO/15-HETE pathway. We found that pulmonary arterial medial thickening caused by hypoxia was alleviated after a treatment of the hypoxic rats with imatinib, which was associated with down-regulations of 15-LO-2 expression and 15-HETE production. Moreover, the increases in cell proliferation and endogenous 15-HETE content by hypoxia were attenuated by the inhibitors of PDGF-β receptor in pulmonary artery smooth muscle cells (PASMCs). The effects of PDGF-BB on cell proliferation and survival were weakened after the administration of 15-LO inhibitors or 15-LO RNA interference. These results suggest that hypoxia promotes PASMCs proliferation and survival, contributing to pulmonary vascular medial hypertrophy, which is likely to be mediated via the PDGF-BB/15-LO-2/15-HETE pathway.     

Application of the ferrous oxidation-xylenol orange assay for the screening of 5-lipoxygenase inhibitors

5-Lipoxygenase (5-LO) is the key enzyme involved in leukotriene synthesis and its improper regulation is implicated in several inflammatory diseases. A rapid and sensitive assay for 5-LO activity suitable for high-throughput format is not yet available. In this study, we examined whether the ferrous oxidation-xylenol orange (FOX) assay could be applicable for the high-throughput screening of 5-LO inhibitors. Using insect cell lysates overexpressing rat 5-LO, the effects of cofactors of 5-LO such as ATP, Ca2+, and L-alpha-phosphatidylcholine (PC) on the color development of FOX reagents were investigated. ATP quenched substantially color development by hydroperoxide, an intermediate of 5-LO reaction, and an optimum concentration of ATP with little interference was determined as 20 microM. Ethylenediaminetetraacetate (0.4 mM) also affected the complex formation with FOX reagents. On the other hand, neither Ca2+ nor PC influenced complex formation with FOX reagents. Under optimized assay conditions, zileuton, a 5-LO-specific inhibitor, exhibited inhibitory potency (IC50 values of 0.1-0.2 microM) similar to that determined by the conventional spectrophotometric assay. Taken together, this study shows that the FOX assay with some modifications can be employed for high-throughput assay format for the measurement of 5-LO activity at the stage of primary screening.     

Phenylsulphonyl urenyl chalcone derivatives as dual inhibitors of cyclo-oxygenase-2 and 5-lipoxygenase

Two series of phenylsulphonyl urenyl chalcone derivatives (UCH) with various patterns of substitution were tested for their effects on nitric oxide (NO) and prostaglandin E2 (PGE2) overproduction in RAW 264.7 macrophages. None of the tested compounds reduced NO production more than 50% at 10 microM but most of them inhibited the generation of PGE2 with IC50 values under the micromolar range. Me-UCH 1, Me-UCH 5, Me-UCH 9, Cl-UCH 1, and Cl-UCH 9 were selected to evaluate their influence on human leukocyte functions and eicosanoids generation. These derivatives selectively inhibited cyclo-oxygenase-2 (COX-2) activity in human monocytes being Me-UCH 5 the most potent (IC50 0.06 microM). Selected compounds also reduced leukotriene B4 synthesis in human neutrophils by a direct inhibition of 5-lipoxygenase (5-LO) activity, with IC50 values from 0.5 to 0.8 microM. In addition, lysosomal enzyme secretion, such as elastase or myeloperoxidase as well as superoxide generation in human neutrophils were also reduced in a similar range. Our findings indicate that UCH derivatives exert a dual inhibitory effect on COX-2/5-LO activity. The profile and potency of these compounds may have relevance for the modulation of the inflammatory and nociceptive responses with reduction of undesirable side-effects associated with NSAIDs.     

12/15-lipoxygenase inhibitors in diabetic nephropathy in the rat

The 12/15-lipoxygenase (12/15-LO) pathway is activated in diabetes mellitus (DM), increasing 12(S)-hydroxyeicosatetraenoic acid (12-HETE). We showed that a 12-LO inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC) inhibited 12/15-LO activity in vivo and assessed the efficacy of another 12/15-LO inhibitor, N-benzyl-N-hydroxy-5-phenylpentamidine (BHPP), to diminish urinary 12-HETE and ameliorate diabetic nephropathy (DN) over 4 months. Rats studied were control (C, n=8), DM (n=6), and rats injected with BHPP (C+BHPP, n=4) and (DM+BHPP, n=5). BHPP 3 mg/kg/day decreased urinary (U) 12-HETE/creatinine (cr) by 30-50% after one injection and after 1 week of daily injections in DM rats. U 12-HETE/cr excretion increased paradoxically in controls given BHPP. There was a highly significant relationship between U 12-HETE/cr excretion and U alb/cr (r=0.79, P<10(-5)), demonstrating that renal 12/15-LO pathway activation is associated with albuminuria. BHPP did not inhibit glomerular collagen synthesis or improve histology. More sustained 12-LO inhibition may improve albuminuria in DN.     

Effect of catechol derivatives on cell growth and lipoxygenase activity

Derivatives of salicylic acid have been synthesized as potential lipoxygenase inhibitors. Agents containing a phenolic dihydroxy moiety showed potent (IC(50)10(-6)-10(-7) M) inhibition of the growth of murine colonic tumour cells in vitro, and were effective inhibitors of 5-, 12- and 15-lipoxygenase in intact cells. The catechols were also potent inhibitors of rabbit reticulocyte 15-lipoxygenase (IC(50) approximately 1 microM).