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 in 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 which did not inhibit NQO1 and were non-toxic to cells. Computational-based molecular modeling simulations demonstrated favorable conformations of indolequinones positioned directly above and in parallel to 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-aminoalkyamino side chain. Hydrogen bonding of the terminal amine nitrogen in the aminoalkylamino side chain was found to be critical for correct orientation of the inhibitors in the active site. These indolequinones were irreversible inhibitors and were found to be at least an 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.

Antitumour indolequinones: synthesis and activity against human pancreatic cancer cells

“…The ability of indolequinones to induce growth inhibition in human cancer cell lines was measured using the MTT assay as previously described. 16,17 …”

“…5–8 In continuation of our studies on the synthesis and biology of indolequinones, we investigated a number of relatively simple tri- and tetra-substituted derivatives initially evaluating their cytotoxicity and also their metabolism by quinone reductase enzymes. 9–16 …”

Hydrazines as versatile chemical biology probes and drug-discovery tools for cofactor-dependent enzymes

“…By the same mechanism, related small molecules inhibit other enzymes with noncovalent FAD cofactors (e.g. NQO2 35 and KDM1A 36 ). Reaction of 3 by an analogous mechanism (Fig.3a, upper) would not, in our experiments, lead to enrichment of an enzyme bearing a noncovalently bound flavin, because covalency with the protein is required for retention on the resin and detection by SDS-PAGE.…”

c-Myc Metabolic Addiction in Cancers Counteracted by Resveratrol and NQO2

“…NQO2 stabilizes C/EBPα degradation mediated by 20S proteasomes [35]. We previously showed that NQO2 is a high affinity target protein of resveratrol: NQO2 binds resveratrol with K D ≤ 50 nM [36]; X-ray crystal analysis shows that binding to resveratrol occurs in a hydrophobic pocket located between dimeric NQO2, possibly where the cosubstrate NRH binds [37]. Since the plasma concentration of resveratrol in humans can reach from 0.5 μM [38] to as high as 4 μM [39,40], it may be suggested that in vivo levels of resveratrol are sufficient for binding and inhibiting NQO2 enzyme activity and modulation of its other functions.…”