Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency

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• 作者：Michelle Armstrong^a ; Christopher van Hoorebeke^a ; Thomas Horn^a ; ^&dagger ; ; Joshua Deschamps^a ; J. Cody Freedman^a ; Chakrapani Kalyanaraman^b ; Matthew P. Jacobson^b ; Theodore Holman^a ; ^{holman@ucsc.edu" class="auth_mail" title="E-mail the corresponding author}
• 关键词：LOX ; lipoxygenase ; h15-LOX-1 ; human reticulocyte 15-lipoxygenase-1 ; tPA ; tissue plasminogen activator ; FDA ; Food and Drug Administration ; s15-LOX-1 ; soybean 15-lipoxygenase-1 ; r15-LOX ; rabbit reticulocyte 15-LOX ; AA ; arachidonic acid ; 12-HpETE ; 12-hydroperoxyeicosatetraenoic acid ; LA ; linoleic acid ; 9R-HpODE ; 9-(R)-hydroperoxyoctadecadienoic acid ; 13-HpODE ; 13-(S)-hydroperoxyoctadecadienoic acid ; 15-HpETE ; 15-hydroperoxyeicosatetraenoic acid ; ICP-MS ; inductively coupled plasma mass spectrometer
• 刊名：Bioorganic & Medicinal Chemistry
• 出版年：2016
• 出版时间：1 November 2016
• 年：2016
• 卷：24
• 期：21
• 页码：5380-5387
• 全文大小：2401 K

文摘

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. IC₅₀ values 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.