title: Indolequinone inhibitors of NRH:quinone oxidoreductase 2. Characterization of the mechanism of inhibition in both cell-free and cellular systems.
abstract: We describe a series of indolequinones as efficient mechanism-based inhibitors of NRH:quinone oxidoreductase 2 (NQO2) for use either in cellular or cell-free systems. Compounds were designed to be reduced in the active site of the enzyme leading to loss of a substituted phenol leaving group and generation of a reactive iminium electrophile. Inhibition of NQO2 activity was assessed in both cell-free systems and the human leukemia K562 cell line. Inhibition of recombinant human NQO2 by the indolequinones was NRH-dependent, with kinetic parameters characteristic of mechanism-based inhibition and partition ratios as low as 2.0. Indolequinones inhibited NQO2 activity in K562 cells at nanomolar concentrations that did not inhibit NQO1 and were nontoxic to cells. Computation-based molecular modeling simulations demonstrated favorable conformations of indolequinones positioned directly above and in parallel with the isoalloxazine ring of FAD, and mass spectrometry extended our previous finding of adduction of the FAD in the active site of NQO2 by an indolequinone-derived iminium electrophile to the wider series of indolequinone inhibitors. Modeling combined with biochemical testing identified key structural parameters for effective inhibition, including a 5-aminoalkylamino side chain. Hydrogen bonding of the terminal amine nitrogen in the aminoalkylamino side chain was found to be critical for the correct orientation of the inhibitors in the active site. These indolequinones were irreversible inhibitors and were found to be at least 1 order of magnitude more potent than any previously documented competitive inhibitors of NQO2 and represent the first mechanism-based inhibitors of NQO2 to be characterized in cellular systems.
authors: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Aurora, Colorado 80045, USA.
authors: Chao
authors: C
authors: Yan
authors: Marine
authors: M
authors: Dufour
authors: David
authors: D
authors: Siegel
authors: Philip
authors: P
authors: Reigan
authors: Joe
authors: J
authors: Gomez
authors: Biehuoy
authors: B
authors: Shieh
authors: Christopher J
authors: CJ
authors: Moody
authors: David
authors: D
authors: Ross
mesh_terms: D000478:Alkylation; D000586:Amination; D001667:Binding, Competitive; D020134:Catalytic Domain; D002474:Cell-Free System / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D006801:Humans; D045563:Indolequinones / Q000737:chemistry* / Q000494:pharmacology; D020014:K562 Cells; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors / Q000737:chemistry; D011808:Quinone Reductases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D045563:Indolequinones; D011994:Recombinant Proteins; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; C419642:NRH - quinone oxidoreductase2; D011808:Quinone Reductases