N-Substituted 5-(1H-Indol-2-yl)-2-methoxyanilines Are Allosteric Inhibitors of the Linoleate Oxygenase Activity of Selected Mammalian ALOX15 Orthologs: Mechanism of Action

Abstract

Here, we describe the first systematic study on the mechanism of substrate-selective inhibition of mammalian ALOX15 orthologs. For this purpose, we prepared a series of N-substituted 5-(1H-indol-2-yl)­anilines and found that (N-(5-(1H-indol-2-yl)-2-methoxyphenyl)­sulfamoyl)­carbamates and their monofluorinated analogues are potent and selective inhibitors of the linoleate oxygenase activity of rabbit and human ALOX15. Introduction of a 2-methoxyaniline moiety into the core pharmacophore plays a crucial role in substrate-selective inhibition of ALOX15-catalyzed oxygenation of linoleic acid at submicromolar concentrations without affecting arachidonic acid oxygenation. Steady-state kinetics, mutagenesis studies, and molecular dynamics (MD) simulations suggested an allosteric mechanism of action. Using a dimer model of ALOX15, our MD simulations suggest that the binding of the inhibitor at the active site of one monomer induces conformational alterations in the other monomer so that the formation of a productive enzyme–linoleic acid complex is energetically compromised.

Different Structures—Similar Effect: Do Substituted 5-(4-Methoxyphenyl)-1H-indoles and 5-(4-Methoxyphenyl)-1H-imidazoles Represent a Common Pharmacophore for Substrate Selective Inhibition of Linoleate Oxygenase Activity of ALOX15?

“…To test the validity of our hypothesis, the octyl (N-(5-(1 H -indol-2-yl)-2-methoxyphenyl)sulfamoyl)carbamate ( 1 ) was prepared according to the procedure we described previously [ 27 ]. N-(2-(5-(4-methoxyphenyl)-2-(pyridin-2-yl)-1 H -imidazol-4-yl)ethyl)-4-pentylbenzenesulfonamide ( 2 ) was synthesized from 4-chloro-1-(4-methoxyphenyl)butan-1-one ( 4 ) according to Weinstein et al [ 21 ], following a slightly modified synthetic protocol (see Supporting Information ).…”

“…This result suggested that the pocket of monomer A needs structural rearrangement to accommodate compound 2 in the substrate-binding pocket. For this reason, additional docking calculations were carried out with compound 2 using the binding cavity of monomer A, once adapted to accommodate compound 1 [ 27 ]. In this reorganized structure of the protein, several binding modes of compound 2 were found.…”

“…Similar conformational differences were recently observed in human ALOX12 dimer using cryo-electron microscopy [ 31 ]. Site-directed mutagenesis of key amino acids that contribute to ALOX15 inter–monomer interaction, stability assays, molecular dynamics (MD) simulations and kinetic modelling allowed extending conventional model of lipoxygenase catalysis by the following elements: (i) in aqueous solutions, ALOX15 may be present as transient dimers; (ii) effectors (inhibitors or activators) may be bound at the substrate-binding pocket of one monomer and may induce conformational changes in the structure of other monomer that bind fatty acid substrates; and (iii) the alterations in the structure of the catalytic monomer modify the mode of substrate alignment at the active site and, thus, modulate the catalytic activity of the enzyme [ 27 , 30 ].…”

“…In a previous study, we found that the 2-arylindole scaffold 1 with its MeO-group attached in the paraposition of the benzene ring ( Figure 1 ) represents an “allosteric determinant” required for selective inhibition of the linoleate oxygenase activity of ALOX15 [ 27 ]. In contrast, the ester group of the sulphocarbamate moiety may be considered an “affinity determinant” required for effective binding of the inhibitor at the enzyme.…”

Identification of novel candidates for inhibition ofLasR, a quorum-sensing receptor of multidrug resistantPseudomonas aeruginosa, through a specialized multi-levelin silicoapproach

“…This is a typical cut-off value for studies with MD simulations followed by MM/GBSA calculations. 21,22,[69][70][71][72][73][74] SHAKE algorithm was used to constrain bonds with hydrogen atoms, enabling the application of an integration time-step of 2 fs.…”