Antioxidative enzymes

NAD(P)H quinone oxidoreductase 1 (NQO1; DT-diaphorase) is a broad specificity enzyme which reduces a range of substrate including quinones.  We are seeking to understand how this protein works and how variant forms of this predispose people to cancer.  Our studies focus on the recombinant human enzyme and combine biochemical, enzymological, biophysical and in silico work.  However, we also aspire to generate yeast “models” to enable us to study variants under controlled in vivo conditions and we are open to collaborations from colleagues who are studying the enzyme in cell culture or other in vivo systems.  In related work, we are also interested in the related enzyme, NRH quinone oxidoreductase 2 (NQO2), the yeast quinone oxidoreductase Lot6p and the bacterial enzyme MdaB.

Project time frame

 This project commenced in 2006 and will end when we have addressed all the interesting questions.

 

Project aims

 

  • To understand how disease-associated variant forms of galactokinase, galactose 1-phosphate uridylyltransferase and UDP-galactose 4’-epinerase fail to function
  • To undertake in silico studies to identify other variants which may have reduced stability compared to the wild-type
  • To identify small molecular which may act as “pharmacological chaperones” to correct the folding of disease-associated variants
  • To study Leloir pathway enzymes from pathogens and to discover compounds which inhibit these
  • To exploit Leloir pathway enzymes in biocatalysis
  • To extend the approaches pioneered for galactosemia to other inherited metabolic diseases

 

 

Project findings and impact

  • The cancer-associated NQO1 variant P187S is massively destabilised compared to the wild-type protein
  • This variant has a much-reduced affinity for the cofactor, FAD
  • Both Lot6p and NQO2 demonstrate negative cooperativity with respect to inhibitors

Research Team

David Timson

Output

  • Nolan KA, Timson DJ, Stratford IJ & Bryce RA (2006) In silico identification and biochemical characterisation of novel substrates and inhibitors of NQO1.  Bioorganic and Medicinal Chemistry Letters  16, 6246-6254.
  • Nolan KA, Zhao H, Faulder PF, Timson DJ, Siegel D, Ross D, Burke Jr TR, Stratford IJ & Bryce RA (2007) Coumarin-based inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity.  Journal of Medicinal Chemistry.  50, 6316-6325.
  • Megarity CF, Gill JRE, Caraher MC, Nolan KA, Stratford IJ & Timson DJ (2014) The two polymorphic forms of human NRH-quinone oxidoreductase 2 (NQO2) have different biochemical properties.  FEBS Letters 588, 1666-1672.
  • Megarity CF, Looi HK & Timson DJ (2014) The Saccharomyces cerevisiae quinone oxidoreductase Lot6p:  stability, inhibition and cooperativity.  FEMS Yeast Research.  14, 797–807.
  • Pey A, Megarity CF & Timson DJ (2014) FAD binding overcomes defects in activity and stability displayed by cancer-associated variants of human NQO1.  Biochimica et Biophysica Acta – Molecular Basis of Disease.  1842, 2163-2173
  • Medina-Carmona E, Palomino-Morales RJ, Fuchs JE, Padín-Gonzalez E, Salido E, Mesa-Torres N Timson DJ & Pey AL (2016) Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands.  Scientific Reports.  6:20331.
  • Pey A, Megarity CF, Medina-Carmona E & Timson DJ (2016) Natural small molecules as stabilizers and activators of cancer-associated NQO1 polymorphisms. Current Drug Targets. 17, 1506-1514.
  • Timson DJ (2017) Dicoumarol:  a drug which hits at least two very different targets in vitamin K metabolism.  Current Drug Targets.  18, 500-510.
  • Medina-Carmona E, Neira JL, Salido E, Fuchs JE, Palomino-Morales R, Timson DJ & Pey AL (2017) Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism.  Scientific Reports.  7:44532.
  • Medina-Carmona E, Fuchs JE, Gavira JA, Muñoz IG, Mesa-Torres N, Neira JL, Morel B, Salido E, Palomino-Morales R, Burgos M, Timson DJ& Pey AL (2017) Vulnerability of human proteins towards disease-associated inactivation through divergent evolution  Human Molecular Genetics.  26, 3531-3544.
  • Betancor I, Timson DJ, Salido E & Pey AL (2018) Natural (and unnatural) small molecules as pharmacological chaperones and inhibitors in cancer. In:  Opportunities for targeting trafficking in drug design, Handbook of Experimental Pharmacology.  In press.

Partners

Dr Angel Pey, University of Grenada

Prof Ian Stratford, University of Manchester

Dr Richard Bryce, University of Manchester

Dr Clare Megarity, University of Oxford