Pharmacological inhibitors of NAD(P)H quinone oxidoreductase, NQO1: Structure/activity relationships and functional activity in tumour cells Karen Ann Nolan a , Katherine Ann Scott a , John Barnes a,b , Jeremy Doncaster b , Roger Clive Whitehead b , Ian James Stratford a, * a School of Pharmacy and Pharmaceutical Sciences, University of Manchester and Manchester Cancer Research, Manchester, Oxford Road, Manchester, M13 9PT, UK b School of Chemistry, University of Manchester, Oxford Road, Manchester, M13 9PT, UK 1. Introduction NAD(P)H quinone oxidoreductase (NQO1, DT-diaphorase, quinone reductase 1, vitamin K reductase, E.C.1.6.99.2) is a ubiquitous, homodimeric flavoprotein with one molecule of FAD non-covalently bound per monomer [1]. NQO1 is generally regarded as a detoxifying enzyme, where its major role has been considered to be its ability to catalyse the reduction of various quinones [2]. NQO1 has also been shown to act as a chaperone, thereby stabilising various proteins, including the tumour suppressor protein p53 [3] and other short-lived proteins such as ornithine decarboxylase [4]. This stabilisation phenomenon is NAD(P)H dependent [5,6], which suggests that the binding of NQO1 to its client protein(s) is most efficient when the enzyme- bound FAD is in its reduced form. Dicoumarol is the most commonly used inhibitor of NQO1, and it acts through competitive binding with NAD(P)H and thereby prevents the two-electron transfer to FAD from occurring. Hence, addition of dicoumarol to cells has been shown to result in the targeting of p53 for degradation [7]. The pharmacological action of dicoumarol for interfering with the function of NQO1 is compromised by extensive protein binding [8] and confounded by ‘‘off-target’’ effects such as the increased production of intracellular superoxide [9] and mitochondrial uncoupling [10]. Thus, we have sought to develop novel inhibitors of NQO1 which retain the inhibitory potency of dicoumarol but lack its unfavourable off-target effects. To achieve this the National Cancer Institute compound database was mined to identify ‘‘dicoumarol-like’’ molecules that could act as competitive inhibitors of NQO1 [11]. From these studies, a computational and synthetic platform was generated to identify novel coumarin- based competitive NQO1 inhibitors, with the aim of discovering agents with potentially superior pharmacological properties to dicoumarol [12]. Previously, NQO1 has been used as the target enzyme in tumour cells to exemplify the ‘‘enzyme directed’’ approach to anticancer drug development [13]. Arguably, the best example of this was in the development of EO9 (5-aziridinyl-3-hydroxymethyl-2-(3- hydroxyprop-1-enyl)-1-methylindole-4,7-dione, Apaziquone). Early work clearly demonstrated a relationship between the toxicity of EO9 and intracellular activity of NQO1 [14–16]. The function of NQO1 was to metabolically activate EO9 to generate Biochemical Pharmacology 80 (2010) 977–981 ARTICLE INFO Article history: Received 17 May 2010 Accepted 14 June 2010 Keywords: NQO1 Dicoumarol Coumarins EO9 p53 ABSTRACT NAD(P)H quinone oxidoreductase (NQO1) has multiple functions in the cell including an ability to act as a detoxifying enzyme and as a protein chaperone. The latter property is particularly important in oncology as one of the client proteins of NQO1 is p53. The inhibitor, dicoumarol, is classically used to probe the biological properties of NQO1, but interpretation of enzyme function is compromised by the multiple ‘‘off-target’’ effects of this agent. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of recombinant human NQO1 have been identified (Nolan et al., J Med Chem 2009;52:7142–56) The purpose of the work reported here is to demonstrate the functional activity of these agents for inhibiting NQO1 in cells. To do this, advantage was taken of the NQO1-mediated toxicity of the chemotherapeutic drug EO9 (Apaziquone). The toxicity of this drug is substantially reduced when the function of NQO1 is inhibited and many of the coumarin-based compounds are more efficient than dicoumarol for inhibiting EO9 toxicity. The ability to do this appears to be related to their capacity to inhibit NQO1 in cell free systems. In conclusion, agents have been identified that may be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues. ß 2010 Elsevier Inc. All rights reserved. * Corresponding author at: The University of Manchester, School of Pharmacy and Pharmaceutical Sciences, G.122 Stopford Building, Oxford Road, Manchester, M13 9PT, UK. Tel.: +44 0 161 275 5634; fax: +44 0 161 275 8342. E-mail address: ian.stratford@manchester.ac.uk (I.J. Stratford). Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm 0006-2952/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2010.06.024