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

Clare F Megarity1, David J Timson2,3

  • 1School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, U.K.

Bioscience Reports
|August 22, 2019
PubMed

Insights

Cancer-associated polymorphisms in human NAD(P)H quinone oxidoreductase 1 (NQO1) do not alter its negative cooperativity with inhibitors. Resveratrol inhibits NQO1, distinct from its effects on NQO2.

Area of Science:

  • Biochemistry
  • Enzymology
  • Molecular Biology

Background:

  • Human NAD(P)H quinone oxidoreductase 1 (NQO1), also known as DT-diaphorase, plays a crucial role in cellular redox homeostasis.
  • NQO1 exhibits negative cooperativity with its inhibitor dicoumarol, a phenomenon that can influence drug efficacy.
  • Two cancer-associated polymorphisms, p.R139W and p.P187S, have been identified in NQO1, but their impact on inhibitor binding and enzyme cooperativity remains unclear.

Purpose of the Study:

  • To investigate whether cancer-associated polymorphisms in NQO1 affect its interaction with inhibitors, specifically dicoumarol.
  • To determine if these polymorphisms alter the negative cooperativity of NQO1 towards dicoumarol.
  • To explore the inhibitory effects of resveratrol and nicotinamide on NQO1 and compare them to NQO2.

Main Methods:

  • Site-directed mutagenesis was used to create NQO1 variants with p.R139W and p.P187S polymorphisms.
  • Enzyme kinetics and binding assays were performed to assess dicoumarol affinity and cooperativity (Hill coefficient).
  • Thermal denaturation studies and inhibition assays were conducted with resveratrol and nicotinamide.

Main Results:

  • Dicoumarol stabilized both NQO1 variants and exhibited higher affinity for p.R139W than p.P187S.
  • Both NQO1 variants displayed negative cooperativity towards dicoumarol, with Hill coefficients similar to the wild-type enzyme (h ≈ 0.5).
  • Resveratrol inhibited NQO1, albeit ~10,000-fold weaker than its inhibition of NQO2, and induced modest negative cooperativity (h = 0.85). Nicotinamide showed non-cooperative inhibition.
  • Nicotinamide stabilized wild-type and p.R139W NQO1, while resveratrol destabilized all tested NQO1 forms.

Conclusions:

  • The cancer-associated NQO1 polymorphisms (p.R139W and p.P187S) do not appear to mediate their effects by altering the enzyme's negative cooperativity towards dicoumarol.
  • Resveratrol demonstrates inhibitory activity against NQO1, independent of its known effects on NQO2, suggesting a potential role in molecular pathology.
  • The differential effects of nicotinamide and resveratrol on NQO1 stability and cooperativity highlight the complex regulatory mechanisms of this enzyme.
Keywords:
DT-diaphoraseNAD(P)H quinone oxidoreductasecancer-associated polymorphismdicoumarolnegative cooperativityresveratrol

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