Discovery of platelet-type 12-human lipoxygenase selective inhibitors by high-throughput screening of structurally diverse libraries

Deschamps, Joshua D.; Gautschi, Jeffrey T.; Whitman, Stephanie; Johnson, Tyler A.; Gassner, N.C.; Crews, Phillip; Holman, Theodore R.

doi:10.1016/j.bmc.2007.08.015

Cited by 59 publications

(49 citation statements)

References 55 publications

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“…Briefly, these two enzymes contained hexa-His tags, and were purified in one step by Ni 2+ affinity chromatography. Epithelial 15-hLO-2 did not contain a His-tag and was purified as previously reported with ion exchange chromatography (36). All enzymes were frozen at −80 °C, with glycerol added (20 % for 12-hLO and 10 % for 15-hLO-1 and 15-hLO-2) to prevent inactivation.…”

Section: Expression and Purification Of Lipoxygenasesmentioning

confidence: 99%

Isotope Sensitive Branching and Kinetic Isotope Effects in the Reaction of Deuterated Arachidonic Acids with Human 12- and 15-Lipoxygenases

et al. 2008

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Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30−80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA, but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2. Keywordsisotope sensitive branching; lipoxygenase; arachidonic acid Lipoxygenases (LOs) are non-heme iron proteins that catalyze the hydrogen atom abstraction from a bisallylic position in polyunsaturated fatty acids and the subsequent addition of molecular oxygen to the resulting substrate radical to generate hydroperoxide products (1). Mammalian LOs catalyze key steps in the conversion of arachidonic acid (AA), 1 a C20 tetraunsaturated fatty acid (20:4, ω−6), to lipoxins and leukotrienes (2). They have been implicated in a number of pathologies including cancer (3,4), atherosclerosis (5) and Alzheimer's disease (6,7). In plants, LOs convert linoleic acid (LA), a C18 bisunsaturated fatty acid (18:2, ω−6), into 13-hydroperoxyoctadecadienoic acid (13-HPODE), a precursor for † This work was supported by the National Institutes of Health (GM44911, WAV and GM56062, TRH) and American Heart Association pre-doctoral fellowships (0615604Z, CJ and 0310006Z, CMM).vddonk@uiuc.edu. tholman@chemistry.ucsc.edu. Supporting information Synthetic procedures for 10,10,13,13-d 4 arachidonic acid and HPLC traces displaying product distributions of the human lipoxygenase reactions are available free of charge via the internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 July 8. Published in final edited form as:Biochemistry. jasmonates and aldehydes involved in signaling, germination, and senescence (8). In mammals, the isozymes are named according to the position of AA that reacts with molecular oxygen (Figure 1). Several human LOs (5-, 12-, and 15-hLOs) have thus far been identified (9), with this study focusing on the latter two groups. Two isozymes of 15-hLO have been characterized in humans and are called 15-hLO-1 (also termed 12/15-hLO) (10,11) and 15-hLO-2 (12). The former exhibits a product selectivity of 15-HPETE over 12-HPETE of about a 9:1 (13), whereas the lat...

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Section: Expression and Purification Of Lipoxygenasesmentioning

confidence: 99%

Isotope Sensitive Branching and Kinetic Isotope Effects in the Reaction of Deuterated Arachidonic Acids with Human 12- and 15-Lipoxygenases

et al. 2008

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“…[15][16][17][18] In an effort to develop small molecules against 5-LOX, chemicals bearing oxazole scaffolds as a bioisostere of thiazole were explored from chemical libraries deposited at Korea Research Institute of Chemical Technology (KRICT, Daejeon, South Korea) using an enzymatic screening assay as described previously. 19,20) As a result, we identified N-aryl-5-aryloxazol-2-amine derivatives that could inhibit 5-LOX catalytic activity potently. Accordingly, a series of N-aryl-5-aryloxazol-2-amine derivatives was synthesized, and tested in an in vitro enzymatic assay to determine the structure-activity relationship (SAR) of those novel 5-LOX inhibitors.…”

Section: )mentioning

confidence: 98%

“…(d , J=8.4 Hz, 1H), 7.78 (s, 1H), 9.11 (s, 1H), 10.04 (s, 1H); 5-(4-Fluorophenyl)oxazol-2-yl) 5-(2,4-Difluorophenyl)oxazol-2-yl) 5-(4-Fluorophenyl)oxazol-2-yl) 5-(4-Fluorophenyl)oxazol-2-yl) 5-(3,4-Dichlorophenyl)oxazol-2-yl) 5-(3-Nitrophenyl)oxazol-2-yl) In Vitro 5-LOX Assay A 5-LOX enzyme assay was carried out with some modifications of the ferric oxidation of xylenol orange (FOX) assay, which is based on the complex formation of Fe 3+ /xylenol orange with absorption at visible wavelengths 19,20). The enzyme sources were prepared from insect cell (Sf21) lysates highly expressing rat 5-LOX as described previously.…”

Section: -((mentioning

confidence: 99%

Synthesis and Biological Evaluation of <i>N</i>-Aryl-5-aryloxazol-2-amine Derivatives as 5-Lipoxygenase Inhibitors

Suh

Yum

Cho

2015

CHEMICAL & PHARMACEUTICAL BULLETIN

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We describe the synthesis and biological evaluation of N-aryl-5-aryloxazol-2-amine derivatives that are able to inhibit 5-lipoxygenase (5-LOX), a key enzyme of leukotriene synthesis, for the treatment of inflammation-related diseases including asthma and rheumatoid arthritis. A novel structural moiety containing oxazole was initially identified from a chemical library using an in vitro enzymatic and cell-based assay, and its synthesized oxazole derivatives were further examined to develop a structure-activity relationship (SAR). SAR analysis demonstrated that a hydroxyl or amino group at the p-position on N-phenyl was essential for the 5-LOX-inhibitory activities of the derivatives, and that other halogen and methyl group-substituted derivatives affected the potency, positively or negatively. As a result, derivatives selected through first-round screening were further optimized using a cell-based assay and an in vivo assay to develop a potent, selective 5-LOX inhibitor. A final hit exhibited an improved efficacy in arachidonic acid-induced ear edema when applied topically but not orally. Moreover, it showed the additional advantage of sustainable antiinflammatory activity over a reference compound, zileuton. Taken together, chemical entities bearing an oxazole scaffold could be promising as therapeutic drugs for the treatment of chronic inflammatory skin disorders.Key words 5-lipoxygenase; antiinflammatory activity; oxazol-2-amine; leukotriene In response to external stresses, several enzymes are involved in the production of inflammatory chemical mediators from arachidonic acid (AA) that is released from the cytoplasmic membrane.1) Specifically, prostaglandins (PGs) and leukotrienes (LTs) are, respectively, formed from an AA substrate, catalyzed by two individual enzymes, cyclooxygenase (COX) and lipoxygenase (LOX), at the bifurcation of the metabolic pathway.2,3) Initial approaches targeting COX-2 selectively in an effort to develop anti-inflammatory drugs proved to be inappropriate due to their adverse cardiac effects. 4) Alternative ways to control inflammatory mediators have been pursued for the treatment of inflammation-related diseases. The 5-lipoxygenase (5-LOX) is a key enzyme in the first step of leukotriene (LT) synthesis, and its dysregulation causes various inflammatory diseases, such as atopy and asthma.5,6) Among the LTs, leukotriene B4 (LTB4) is a key chemoattractant for neutrophils recruitment, a hallmark of acute inflammation 7) and the others, termed collectively the "cysteinyl LTs," act as the slowly reacting substances of anaphylaxis. 8)Thus, 5-LOX inhibitors that are able to block LTs production could be effective therapeutic agents to treat inflammatory diseases. Presently, only zileuton has been approved by the Food and Drug Administration (FDA) as a direct 5-LOX inhibitor. 9) However, there are many anti-inflammatory drugs based on the abrogation of the ligand-receptor interaction or indirect interference in the process of 5-LOX activation. [10][11][12] Recently, there has been special i...

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“…30) Such target proteins include fatty acid synthase, 31) cytochrome P450s, 32) 12-lipoxygenase, 33) acidic sphingomyelinase, 34) Ca 2+ -ATPase, 35) aromatase, 36) human immunodeficiency virus (HIV)-1 protease, 37) Ca…”

Section: Inhibitory Potency and Selectivity Of Mangostinsmentioning

confidence: 99%

“…40) The IC 50 values of α-mangostin and γ-mangostin for the known target enzymes are 0.58-33 µM [31][32][33][34][35][36][37][38] and 0.8-10 µM, [36][37][38][39][40] respectively, which are higher than those of the two mangostins for AKR1B10. To our knowledge, the IC 50 value (0.018 µM) of γ-mangostin for AKR1B10 is the lowest among those of the two mangostins for other enzymes reported to date.…”

Section: +mentioning

confidence: 99%

Inhibition of Human Aldose Reductase-Like Protein (AKR1B10) by α- and γ-Mangostins, Major Components of Pericarps of Mangosteen

Soda

Endo

Matsunaga

et al. 2012

Biological & Pharmaceutical Bulletin

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A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, was recently identified as both diagnostic marker and therapeutic target in the treatment of several types of cancer. In this study, we have examined AKR1B10 inhibition by five xanthone derivatives, components of pericarps of mangosteen, of which α-and γ-mangostins show potential anti-cancer properties. Among the five xanthones, γ-mangostin was found to be the most potent competitive inhibitor (inhibition constant, 5.6 nM), but its 7-methoxy derivative, α-mangostin, was the second potent inhibitor (inhibition constant, 80 nM). Molecular docking of the two mangostins in AKR1B10 and site-directed mutagenesis of the putative binding residues revealed that Phe123, Trp220, Val301 and Gln303 are important for the tight binding of γ-mangostin, and suggested that the 7-methoxy group of α-mangostin impairs the inhibitory potency by altering the orientation of the inhibitor molecule in the substrate-binding site of the enzyme.Key words aldo-keto reductase 1B10; aldose reductase; mangostin; xanthone; anti-cancer property; molecular docking A human member of the aldo-keto reductase (AKR) superfamily, AKR1B10, is a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase, which was originally identified as a human aldose reductase (AKR1B1)-like protein that is up-regulated in hepatocellular carcinomas. 1)Over-expression of AKR1B10 has been also observed in other tumors, such as smokers' non-small cell lung carcinomas, 2) uterine carcinomas, 3) cholangiocarcinomas, 4) pancreatic carcinoma, 5) and breast cancer.6) The silencing of the AKR1B10 gene results in growth inhibition of cancer cells [5][6][7][8] and hepatocellular carcinoma xenografts in mice, 9) and its elevated expression in turn promotes proliferation of cancer cells, 10,11) indicating that the enzyme participates in tumor development. Due to its high catalytic efficiency towards aliphatic aldehydes, retinals and isoprenyl aldehydes, 1,[6][7][8]12,13) the roles suggested for AKR1B10 in cell carcinogenesis and tumor development are detoxification of cytotoxic carbonyls derived from lipid peroxidation, [6][7][8] decrease in retinoic acid synthesis, 12) and modulation of protein prenylation. 6,11,13) In addition, AKR1B10 is suggested to promote fatty acid synthesis in liver cancer cells by blocking the ubiquitin-dependent degradation of acetyl CoA carboxylase. 8) Thus, this enzyme has been recognized not only as a potential diagnostic and/or prognostic marker, but also as a potential therapeutic target for the prevention and treatment of the above types of cancer. However, because of the high structural similarity between AKR1B10 and AKR1B1 that plays distinct roles in glucose and prostaglandin metabolism, 14,15) the selective inhibition of AKR1B10 over AKR1B1 is required for the development of treatments targeting the types of cancer linked to AKR1B10.Synthetic and natural compounds that show inhibitory effects on AKR1B10 have been reported, as reviewed by Matsunaga et al. 1...

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Discovery of platelet-type 12-human lipoxygenase selective inhibitors by high-throughput screening of structurally diverse libraries

Cited by 59 publications

References 55 publications

Isotope Sensitive Branching and Kinetic Isotope Effects in the Reaction of Deuterated Arachidonic Acids with Human 12- and 15-Lipoxygenases

Isotope Sensitive Branching and Kinetic Isotope Effects in the Reaction of Deuterated Arachidonic Acids with Human 12- and 15-Lipoxygenases

Synthesis and Biological Evaluation of <i>N</i>-Aryl-5-aryloxazol-2-amine Derivatives as 5-Lipoxygenase Inhibitors

Inhibition of Human Aldose Reductase-Like Protein (AKR1B10) by α- and γ-Mangostins, Major Components of Pericarps of Mangosteen

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