Inhibitory and mechanistic investigations of oxo-lipids with human lipoxygenase isozymes

Oxo-lipids, a large family of oxidized human lipoxygenase (hLOX) products, are of increasing interest to researchers due to their involvement in different inflammatory responses in the cell. Oxo-lipids are unique because they contain electrophilic sites that can potentially form covalent bonds through a Michael addition mechanism with nucleophilic residues in protein active sites and thus increase inhibitor potency. Due to the resemblance of oxo-lipids to LOX substrates, the inhibitor potency of 4 different oxo-lipids; 5-oxo-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-oxo-ETE), 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE), 12-oxo-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid (12-oxo-ETE), and 13-oxo-9,11-(Z,E)-octadecadienoic acid (13-oxo-ODE) were determined against a library of LOX isozymes; leukocyte 5-lipoxygenase (h5-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), human platelet 12-lipoxygenase (h12-LOX), human epithelial 15-lipoxygenase-2 (h15-LOX-2), soybean 15-lipoxygenase-1 (s15-LOX-1), and rabbit reticulocyte 15-LOX (r15-LOX). 15-Oxo-ETE exhibited the highest potency against h12-LOX, with an IC₅₀ = 1 ± 0.1 μM and was highly selective. Steady state inhibition kinetic experiments determined 15-oxo-ETE to be a mixed inhibitor against h12-LOX, with a K_ic value of 0.087 ± 0.008 μM and a K_iu value of 2.10 ± 0.8 μM. Time-dependent studies demonstrated irreversible inhibition with 12-oxo-ETE and h15-LOX-1, however, the concentration of 12-oxo-ETE required (K_i = 36.8 ± 13.2 μM) and the time frame (k₂ = 0.0019 ± 0.00032 s⁻¹) were not biologically relevant. These data are the first observations that oxo-lipids can inhibit LOX isozymes and may be another mechanism in which LOX products regulate LOX activity.     

Inducible expression of 15-lipoxygenase-2 and 8-lipoxygenase inhibits cell growth via common signaling pathways

Human 15-lipoxygenase (LOX)-2 and mouse 8-LOX represent orthologous members of the LOX family but display different positional specificities and tissue distribution. To study the functional role of 15-LOX-2 and 8-LOX in keratinocytes, an inducible Tet-On gene expression system was established in the premalignant mouse keratinocyte cell line 308. Doxycycline (dox)-induced expression of enzymatically active 15-LOX-2 and 8-LOX led to an inhibition of cell growth that was associated with an inhibition of DNA synthesis, as shown by a 15-46% reduction of 5-bromo-2-deoxy-uridine (BrdU) incorporation. The inhibitory effects were increased in the presence of exogenous arachidonic acid. In contrast, addition of linoleic acid or the LOX inhibitor baicalein reversed the growth-inhibitory effects. Treatment of the cells with 15-hydroxyeicosatetraenoic acid (HETE) or 8-HETE resulted in a similar inhibition of BrdU incorporation, whereas 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE, in contrast, had no effects. Dox-induced keratinocytes showed increased levels of reactive oxygen species (ROS). The antioxidant N-acetyl-L-cysteine and a specific inhibitor of p38 mitogen-activated protein kinase, but not of extracellular signal-regulated kinase 1/2 or c-Jun N-terminal kinase/stress-activated kinases, completely abolished the LOX-induced growth inhibition, indicating a critical role of ROS and p38. Our data suggest that 15-LOX-2 and 8-LOX, although displaying different positional specificity, may use common signaling pathways to induce growth inhibition in premalignant epithelial cells.     

Discovery of a Novel Dual Fungal CYP51/Human 5-Lipoxygenase Inhibitor: Implications for Anti-Fungal Therapy

We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC₅₀ = 700 nM) and CYP51 (IC₅₀ = 43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.     

A potent and selective inhibitor targeting human and murine 12/15-LOX

Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood–brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC₅₀ potency of 3.4 ± 0.5 μM against h12/15-LOX in vitro and an ex vivo IC₅₀ potency of approximately 10 μM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 μM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a K_ic of 1.0 ± 0.08 μM and a K_iu of 6.0 ± 3.3 μM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.     

Pseudoperoxidase investigations of hydroperoxides and inhibitors with human lipoxygenases

Understanding the mode of action for lipoxygenase (LOX) inhibitors is critical to determining their efficacy in the cell. The pseudoperoxidase assay is an important tool for establishing if a LOX inhibitor is reductive in nature, however, there have been difficulties identifying the proper conditions for each of the many human LOX isozymes. In the current paper, both the 234 nM decomposition (UV) and iron-xylenol orange (XO) assays are shown to be effective methods of detecting pseudoperoxidase activity for 5-LOX, 12-LOX, 15-LOX-1 and 15-LOX-2, but only if 13-(S)-HPODE is used as the hydroperoxide substrate. The AA products, 12-(S)-HPETE and 15-(S)-HPETE, are not consistent hydroperoxide substrates since they undergo a competing transformation to the di-HETE products. Utilizing the above conditions, the selective 12-LOX and 15-LOX-1 inhibitors, probes for diabetes, stroke and asthma, are characterized for their inhibitory nature. Interestingly, ascorbic acid also supports the pseudoperoxidase assay, suggesting that it may have a role in maintaining the inactive ferrous form of LOX in the cell. In addition, it is observed that nordihydroguaiaretic acid (NDGA), a known reductive LOX inhibitor, appears to generate radical species during the pseudoperoxidase assay, which are potent inhibitors against the human LOX isozymes, producing a negative pseudoperoxidase result. Therefore, inhibitors that do not support the pseudoperoxidase assay with the human LOX isozymes, should also be investigated for rapid inactivation, to clarify the negative pseudoperoxidase result.     

Synthesis and biological evaluation of a novel class of rofecoxib analogues as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs)

A group of 4-(4-methanesulfonylphenyl)-3-phenyl-2(5H)furanones possessing an acetyl, 3-oxobut-1-ynyl, [hydroxyl(or alkoxy)imino]alkyl, [hydroxyl(or alkoxy)imino]alkynyl, and N-alkoxy(or N-phenoxy)carbonyl-N-hydroxy-N-ethylamino substituents at the para-position of the C-3 phenyl ring of rofecoxib were synthesized. This group of compounds was designed for evaluation as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs) that exhibit in vivo anti-inflammatory and analgesic activities. In vitro COX-1/COX-2, and 5-LOX/15-LOX, isozyme inhibition structure-activity relationships identified 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) having an optimal combination of COX-2 (COX-2 IC50 = 1.4 microM; COX-2 SI > 71), and 5-LOX and 15 LOX (5-LOX IC50 = 0.28 microM; 15-LOX IC50 = 0.32 microM), inhibitory effects. It was also discovered that 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (18a) possesses dual COX-2 (IC50 = 2.7 microM) and 5-LOX (IC50 = 0.30 microM) inhibitor actions. Further in vivo studies employing a rat carrageenan-induced paw edema model showed that the oxime compounds (17a, 18a) were more potent anti-inflammatory agents than the 5-LOX inhibitor caffeic acid, and 15-LOX inhibitor nordihydroguaiaretic acid (NDGA), but less potent than the selective COX-2 inhibitor celecoxib. The results of this investigation showed that incorporation of a para-oxime moiety on the C-3 phenyl ring of rofecoxib provides a suitable template for the design of dual inhibitors of the COX and LOX enzymes.     

Challenging inflammatory process at molecular,cellular and in vivo levels via some new pyrazolyl thiazolones

The work reported herein describes the synthesis of a new series of anti-inflammatory pyrazolyl thiazolones. In addition to COX-2/15-LOX inhibition, these hybrids exerted their anti-inflammatory actions through novel mechanisms. The most active compounds possessed COX-2 inhibitory activities comparable to celecoxib (IC₅₀ values of 0.09–0.14 µM) with significant 15-LOX inhibitory activities (IC₅₀s 1.96 to 3.52 µM). Upon investigation of their in vivo anti-inflammatory activities and ulcerogenic profiles, these compounds showed activity patterns equivalent or more superior to diclofenac and/or celecoxib. Intriguingly, the most active compounds were more effective than diclofenac in suppressing monocyte-to-macrophage differentiation and inflammatory cytokine production by activated macrophages, as well as their ability to induce macrophage apoptosis. The latter finding potentially adds a new dimension to the previously reported anti-inflammatory mechanisms of similar compounds. These compounds were effectively docked into COX-2 and 15-LOX active sites. Also, in silico predictions confirmed the appropriateness of these compounds as drug-like candidates.     

Characteristics of the Inhibition of Potato (Solanum tuberosum) Invertase by an Endogenous Proteinaceous Inhibitor in Potatoes

Effect of several parameters on inhibition of potato (Solanum tuberosum) invertase by its endogenous proteinaceous inhibitor was determined using homogeneous preparations of both proteins. The inhibitor and invertase formed an inactive complex with an observed association rate constant at pH 4.70 and 37°C of 8.82 × 10² per molar per second and a dissociation rate constant of 3.3 × 10⁻³ per minute. The inhibitor appeared to bind to invertase in more than one step. Initial interaction (measured by loss of invertase activity) was rapid, relatively weak, readily reversible (K_i of 2 × 10⁻⁶ molar) and noncompetitive with substrate at pH 4.70. Initial interaction was probably followed by isomerization to a tighter (K_i of 6.23 × 10⁻⁸ molar) complex, which dissociated slowly with a half-time of 3.5 hour. Interaction between enzyme and inhibitor appeared to be of ionic character and essentially pH independent between pH 3.5 and 7.4.     

Biochemical Characterization of CTX-M-15 from Enterobacter cloacae and Designing a Novel Non-β-Lactam-β-Lactamase Inhibitor

The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of bla _CTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5α transformed with bla _CTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC₅₀ value (6 nM), high affinity (K _i = 0.017 µM) and better acylation efficiency (k ₊₂/K′ = 0.44 µM⁻¹s⁻¹). It forms an acyl-enzyme covalent complex, which is quite stable (k ₊₃ = 0.0057 s⁻¹). Since increasing resistance has been reported against conventional β-lactam antibiotic-inhibitor combinations, we aspire to design a non-β-lactam core containing β-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC₅₀ (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (K _i = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-β-lactam containing β-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.     

Synthesis and biological evaluation of 1,3-diphenylprop-2-yn-1-ones as dual inhibitors of cyclooxygenases and lipoxygenases

A new class of 1,3-diphenylprop-2-yn-1-ones possessing a p-MeSO2 COX-2 phamacophore on the C-3 phenyl ring was designed for evaluation as dual inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX). Among the group of compounds evaluated, 1-(4-fluorophenyl)-3-(4-methanesulfonylphenyl)prop-2-yn-1-one (11j) exhibited excellent COX-2 inhibitory potency (COX-2 IC50 = 0.1 microM) and selectivity (SI = 300), whereas 1-(4-cyanophenyl)-3-(4-methanesulfonylphenyl)prop-2-yn-1-one (11d) exhibited an optimal combination of COX and LOX inhibition (COX-2 IC50 = 1.0 microM; COX-2 SI = 31.5; 5-LOX IC50 = 1.0 microM; 15-LOX IC50 = 3.2 microM).     

Synthesis and biological evaluation of N-difluoromethyl-1,2-dihydropyrid-2-one acetic acid regioisomers: Dual Inhibitors of cyclooxygenases and 5-lipoxygenase

A new group of acetic acid (7a–c, R¹ = H), and propionic acid (7d–f, R¹ = Me), regioisomers wherein a N-difluoromethyl-1,2-dihydropyrid-2-one moiety is attached via its C-3, C-4, and C-5 position was synthesized. This group of compounds exhibited a more potent inhibition, and hence selectivity, for the cyclooxygenase-2 (COX-2) relative to the COX-1 isozyme. Attachment of the N-difluoromethyl-1,2-dihydropyrid-2-one ring system to an acetic acid, or propionic acid, moiety confers potent 5-LOX inhibitory activity, that is, absent in traditional arylacetic acid NSAIDs. 2-(1-Difluoromethyl-2-oxo-1,2-dihydropyridin-5-yl)acetic acid (7c) exhibited the best combination of dual COX-2 and 5-LOX inhibitory activities. Molecular modeling (docking) studies showed that the highly electronegative CHF₂ substituent present in 7c, that showed a modest selectivity for the COX-2 isozyme, is oriented within the secondary pocket (Val523) present in COX-2 similar to the sulfonamide (SO₂NH₂) COX-2 pharmacophore present in celecoxib, and that the N-difluoromethyl-1,2-dihydropyrid-2-one pharmacophore is oriented close to the region containing the LOX enzyme catalytic iron (His361, His366, and His545). Accordingly, the N-difluoromethyl-1,2-dihyrdopyrid-2-one moiety possesses properties suitable for the design of dual COX-2/5-LOX inhibitory drugs.     

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.     

Phanerochaete chrysosporium β-Glucosidases: Induction, Cellular Localization, and Physical Characterization

Phanerochaete chrysosporium produces intracellular soluble and particulate β-glucosidases and an extracellular β-glucosidase. The extracellular enzyme is induced by cellulose but repressed in the presence of glucose. The molecular weight of this enzyme is 90,000. The K_m for p-nitrophenyl-β-glucoside is 1.6 × 10⁻⁴ M; the K_i for glucose, a competitive inhibitor, is 5.0 × 10⁻⁴ M. The K_m for cellobiose is 5.3 × 10⁻⁴ M. The intracellular soluble enzyme is induced by cellobiose; this induction is prevented by cycloheximide. The presence of 300 mM glucose in the medium, however, had no effect on induction. The K_m for p-nitrophenyl-β-glucoside is 1.1 × 10⁻⁴ M. The molecular weight of this enzyme is ~410,000. Both enzymes have an optimal temperature of 45°C and an E_act of 9.15 kcal (ca. 3.83 × 10⁴ J). The pH optima, however, were ~7.0 and 5.5 for the intracellular and extracellular enzymes, respectively.     

A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase

Retinal hyperpermeability and subsequent macular edema is a cardinal feature of early diabetic retinopathy (DR). Here, we investigated the role of bioactive lipid metabolites, in particular 12/15-lipoxygenase (LOX)-derived metabolites, in this process. LC/MS lipidomic screen of human retinal endothelial cells (HRECs) demonstrated that 15-HETE was the only significantly increased metabolite (2.4 ± 0.4-fold, P = 0.0004) by high glucose (30 mM) treatment. In the presence of arachidonic acid, additional eicosanoids generated by 12/15-LOX, including 12- and 11-HETEs, were significantly increased. Fluorescein angiography and retinal albumin leakage showed a significant decrease in retinal hyperpermeability in streptozotocin-induced diabetic mice lacking 12/15-LOX compared with diabetic WT mice. Our previous studies demonstrated the potential role of NADPH oxidase in mediating the permeability effect of 12- and 15-HETEs, therefore we tested the impact of intraocular injection of 12-HETE in mice lacking the catalytic subunit of NADPH oxidase (NOX2). The permeability effect of 12-HETE was significantly reduced in NOX2^−/− mice compared with the WT mice. In vitro experiments also showed that 15-HETE induced HREC migration and tube formation in a NOX-dependent manner. Taken together our data suggest that 12/15-LOX is implicated in DR via a NOX-dependent mechanism.     

Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction

Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purified by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic substitution (S_Ni) between the hydroperoxy (nucleophile) and epoxy group (electrophile). Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the autoxidation reactions of polyunsaturated fatty acids.     

Dual Effects of Ketoconazole cis-Enantiomers on CYP3A4 in Human Hepatocytes and HepG2 Cells

Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (−)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (−)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, K_i 0.92 µM for testosterone, IC₅₀ 1.46 µM, K_i 2.52 µM for midazolam; (−)-ketoconazole IC₅₀ 0.90 µM, K_i 0.17 µM for testosterone, IC₅₀ 1.04 µM, K_i 1.51 µM for midazolam).     

Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate

The H⁺-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl⁻ and NO₃⁻. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K_m of the enzyme for ATP but caused relatively little alteration of the V_max. Nitrate increased K_m only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl⁻. Other monovalent anions that specifically increased the K_m of the ATPase for ATP were, in order of increasing K_i, SCN⁻, ClO₄⁻, and ClO₃⁻. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO₃⁻, was a competitive inhibitor of the ATPase but ADP and NO₃⁻ did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO₃⁻ does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO₃⁻ exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl⁻ nor NO₃⁻ are involved in substrate binding.     

15-Lipoxygenase metabolism of 2-arachidonylglycerol. Generation of a peroxisome proliferator-activated receptor alpha agonist

The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. Furthermore, 2-AG treatment of COS-7 cells transiently transfected with human 15-LOX expression vectors or normal human epidermal keratinocytes results in the production and extracellular release of 15-hydroxyeicosatetraenoic acid glyceryl ester (15-HETE-G), establishing that lipoxygenase metabolism of 2-AG occurs in an eukaryotic cellular environment. Investigations into the potential biological actions of 15-HETE-G indicate that this lipid, in contrast to its free-acid counterpart, acts as a peroxisome proliferator-activated receptor alpha agonist. The results demonstrate that 15-LOXs are capable of acting on 2-AG to provide 15-HETE-G and elucidate a potential role for endocannabinoid oxygenation in the generation of peroxisome proliferator-activated receptor alpha agonists.     

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.