Cancer-associated variants of human NQO1: impacts on inhibitor binding and cooperativity

Abstract

Human NAD(P)H quinone oxidoreductase (DT-diaphorase, NQO1) exhibits negative cooperativity towards its potent inhibitor, dicoumarol. Here, we addressed the hypothesis that the effects of the two cancer-associated polymorphisms (p.R139W and p.P187S) may be partly mediated by their effects on inhibitor binding and negative cooperativity. Dicoumarol stabilized both variants and bound with much higher affinity for p.R139W than p.P187S. Both variants exhibited negative cooperativity towards dicoumarol; in both cases, the Hill coefficient (h) was approximately 0.5 and similar to that observed with the wild-type protein. NQO1 was also inhibited by resveratrol and by nicotinamide. Inhibition of NQO1 by resveratrol was approximately 10,000-fold less strong than that observed with the structurally similar enzyme, NRH quinine oxidoreductase 2 (NQO2). The enzyme exhibited non-cooperative behaviour towards nicotinamide, whereas resveratrol induced modest negative cooperativity (h = 0.85). Nicotinamide stabilized wild-type NQO1 and p.R139W towards thermal denaturation but had no detectable effect on p.P187S. Resveratrol destabilized the wild-type enzyme and both cancer-associated variants. Our data suggest that neither polymorphism exerts its effect by changing the enzyme’s ability to exhibit negative cooperativity towards inhibitors. However, it does demonstrate that resveratrol can inhibit NQO1 in addition to this compound’s well-documented effects on NQO2. The implications of these findings for molecular pathology are discussed.

The Catalytic Cycle of the Antioxidant and Cancer-Associated Human NQO1 Enzyme: Hydride Transfer, Conformational Dynamics and Functional Cooperativity

“…We provide here a kinetic analysis of the oxidoreductase cycle of NQO1 in unprecedented detail. One of the main conclusions of our study is the existence of non-equivalent active sites in the protein dimer that differ in activity by about 20-fold and, thus, this could explain previous reports on the negative binding cooperativity towards inhibitors such as Dic [48,49]. At this point, we must note that in a thermodynamic sense, the existence of two active sites with different efficiencies (i.e., non-identical active sites) cannot be distinguished from two active sites displaying negative cooperativity (i.e., identical and non-independent active sites) [32,84].…”

“…Considering previous equilibrium binding and kinetic studies [31,48,49,54], the strong evidence provided in this work for the existence of non-equivalent active sites in the human NQO1 is not striking. Thus, our work helps to reconcile previous binding and steady-kinetic analyses focused on NAD(P)H and Dic.…”

“…However, we observed that about half of the HT process occurred through fast and slow pathways in the presence of Dic (that slowed down both pathways by three orders of magnitude). These results indicate that Dic binding causes a larger kinetic uncoupling between the two active sites, thus contributing to an explanation for the negative, but not extreme, cooperativity observed in inhibition studies (with Hill coefficients of about 0.5, corresponding to a cooperative binding Gibbs energy of about 1.5 kcal•mol −1 ; [48,49]). Interestingly, negative cooperativity between FAD binding sites of a similar magnitude is also observed in the apo-enzyme [54].…”

“…Dic typically in the 1−20 nM range; [33,49,55] and Section 3.2.1). Therefore, these inhibition studies with Dic strongly supported that the reduction of the FAD cofactor at the two active sites of the NQO1 dimer occurs at different rates.…”

Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1

“…Superoxide also produced [167] Benzofuroxans Reduction by NQO1 may play a minor role in cytotoxicity [168] Nitroaromatics Reactivity correlates with electrode potential [169,170] Aminochrome Reaction is important in protection against Parkinson's Disease and other neurological diseases [171,172] Inhibitors Dicoumarol and derivatives thereof High affinity; competes with NAD(P)H; negatively cooperative; often used in experimental studies; derivatives may be anticancer lead compounds; dissociates NQO1-p53 complexes resulting in increased p53 degradation and inhibition of apoptosis [143,151,154,156,173,174] Curcumin May dissociate NQO1-p53 complexes resulting in increased p53 degradation and inhibition of apoptosis. Other studies suggest it may enhance the NQO1-p53 interaction in vivo [175,176] Resveratrol Potent inhibitor of the related protein NQO2; only weakly inhibits NQO1 [177] Warfarin NQO1 is a secondary target for this anticoagulant [174,178] [190,191] Preprints (www.preprints.org) | NOT PEER-REVIEWED Interaction occurs in response to genotoxic stress. [194] Table A2.…”