title: Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416.
abstract: 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.
authors.affiliation: Institute of Pharmaceutical Chemistry/ZAFES, University of Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hörnig
authors.forename: Stavroula
authors.initials: S
authors.lastname: Markoutsa
authors.forename: Ann-Kathrin
authors.initials: AK
authors.lastname: Häfner
authors.forename: Sven
authors.initials: S
authors.lastname: George
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Wisniewska
authors.forename: Carmen B
authors.initials: CB
authors.lastname: Rödl
authors.forename: Bettina
authors.initials: B
authors.lastname: Hofmann
authors.forename: Thorsten
authors.initials: T
authors.lastname: Maier
authors.forename: Michael
authors.initials: M
authors.lastname: Karas
authors.forename: Oliver
authors.initials: O
authors.lastname: Werz
authors.forename: Dieter
authors.initials: D
authors.lastname: Steinhilber
mesh_terms: D000328:Adult; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000378:metabolism*; D016718:Arachidonic Acid / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism*; D004963:Estrenes / Q000737:chemistry / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D016859:Lipoxygenase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D051379:Mice; D008958:Models, Molecular; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D011760:Pyrrolidinones / Q000737:chemistry / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D004963:Estrenes; D016859:Lipoxygenase Inhibitors; D050505:Mutant Proteins; D011760:Pyrrolidinones; D013438:Sulfhydryl Compounds; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C074854:U 73343; D016718:Arachidonic Acid; D001094:Arachidonate 5-Lipoxygenase; D003545:Cysteine