Mechanism‐Based Inhibition of Quinone Reductase 2 (NQO2): Selectivity for NQO2 over NQO1 and Structural Basis for Flavoprotein Inhibition

Abstract

A role for the flavoprotein NRH:quinone oxidoreductase 2 (NQO2, QR2) in human diseases such as malaria, leukemia and neurodegeneration has been proposed. In order to explore the potential of NQO2 as a therapeutic target, we have developed potent and selective mechanism-based inhibitors centered on the indolequinone pharmacophore. The compounds show remarkable selectivity for NQO2 over the closely related flavoprotein NQO1 with small structural changes defining selectivity. Biochemical studies confirmed mechanism-based inhibition while X-ray crystallography and mass spectrometry revealed the nature of the inhibitor interaction with the protein. These indolequinones represent the first mechanism-based inhibitors of NQO2, and their novel mode of action involving alkylation of the flavin cofactor, provides significant advantages over existing competitive inhibitors in terms of potency and irreversibility, and will open new opportunities to define the role of NQO2 in disease.

Indolequinone Inhibitors of NRH:Quinone Oxidoreductase 2. Characterization of the Mechanism of Inhibition in both Cell-Free and Cellular Systems

“…Inhibition of NQO2 by the indolequinones appeared to be irreversible since desalting of the indolequinone-inhibited protein using the Millipore Microcon Molecular Cutoff device did not result in recovery of enzyme activity (data not shown). To determine if these compounds could also inhibit the related enzyme NQO1 we incubated rhNQO1 with indolequinones (1 µM) in the presence and absence of NADH and in these studies no inhibition of NQO1 was detected (27). …”

“…Imatinib mesylate was purchased from LC laboratories (Woburn, MA). The indolequinones 5-(4-aminobutyl)amino-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione ( 1 ), 5-(4-aminobutyl)amino-1,2-dimethyl-3-[(2,4,6-trifluorophenoxy)methyl]indole-4,7-dione ( 2 ), 6-(4-aminobutyl)amino-1,2-dimethyl-3-[(2,4,6-trifluorophenoxy)methyl]indole-4,7-dione ( 3 ), 5-(3-aminopropyl)amino-1,2-dimethyl-3-[(2,4,6-trifluorophenoxy)methyl]indole-4,7-dione ( 4 ), 5-(3-dimethylamino)propylamino-1,2-dimethyl-3-[(2,4,6-trifluorophenoxy)methyl]indole-4,7-di one ( 5 ), 6-(3-dimethylamino)propylamino-1,2-dimethyl-3-[(2,4,6-trifluorophenoxy)methyl]indole-4,7-dione ( 6 ), 5-(3-dimethylamino)propylmethylamino −1,2-dimethyl-3-[(2,4,6-trifluorophenoxy)methyl]indole-4,7-dione ( 7 ), 5-(3-dimethylamino)propylamino-1,2-dimethyl-3-(phenoxymethyl)indole-4,7-dione ( 8 ), and 5-(3-dimethylamino)propylamino-1,2-dimethyl-3-(hydroxymethyl)indole-4,7-dione ( 9 ) were synthesized using published methods (27) except that indolequinones 3 and 6 were prepared as described in the supporting information. Recombinant human NQO1 (rhNQO1) was purified from Escherichia coli using Cibacron blue affinity chromatography as previously described (28).…”

“…All of these studies point to an emerging role for NQO2 in diverse physiological and disease process but one major obstacle in defining the role of NQO2 in complex cellular systems has been the absence of potent and specific inhibitors of the enzyme. We have recently examined the structural requirements for selective inhibition of NQO2 relative to NQO1 (27) and proposed a novel mechanism of inhibition involving flavin adduction. In this study, we have characterized a series of indolequinones as mechanism-based inhibitors of NQO2 that can be utilized in both cell-free and cellular systems.…”

NRH: Quinone Reductase activity results in the accumulation of endogenous dopamine in the retina

“…The kinetics of this 24-kDa cytosolic flavoprotein proceeds via a double displacement mechanism [13] . Through this mechanism, FAD is reduced by the cosubstrate, which then diffuses out from the binding pocket, permitting occupation of the active site by the substrate.…”

“…QR2 is able to doubly reduce quinones such as menadione, a synthetic vitamin K precursor, the coenzymes Q0-10 [11] and anti-cancer pro-drugs such as CB1954 [12] . The kinetics of this 24-kDa cytosolic flavoprotein proceeds via a double displacement mechanism [13] . Through this mechanism, FAD is reduced by the cosubstrate, which then diffuses out from the binding pocket, permitting occupation of the active site by the substrate.…”