title: Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.
abstract: Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, United States.
authors.forename: Michelle
authors.initials: M
authors.lastname: Armstrong
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, United States.
authors.forename: Christopher
authors.initials: C
authors.lastname: van Hoorebeke
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Horn
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, United States.
authors.forename: Joshua
authors.initials: J
authors.lastname: Deschamps
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, United States.
authors.forename: J Cody
authors.initials: JC
authors.lastname: Freedman
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA 94143, United States.
authors.forename: Chakrapani
authors.initials: C
authors.lastname: Kalyanaraman
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA 94143, United States.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Jacobson
authors.affiliation: Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064, United States. Electronic address: holman@ucsc.edu.
authors.forename: Theodore
authors.initials: T
authors.lastname: Holman
mesh_terms: D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D016859:Lipoxygenase Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013329:Structure-Activity Relationship
chemical_list: D016859:Lipoxygenase Inhibitors; D001093:Arachidonate 15-Lipoxygenase
keywords: 15-Lipoxygenase-1; Docking; Human; Inhibitor; Mutagenesis