The synthesis is reported here of two novel series of inhibitors of human NAD(P)H quinone oxidoreductase-1 (NQO1), an enzyme overexpressed in several types of tumor cell. The first series comprises substituted symmetric dicoumarol analogues; the second series contains hybrid compounds where one 4-hydroxycoumarin system is replaced by a different aromatic moiety. Several compounds show equivalent or improved NQO1 inhibition over dicoumarol, both in the presence and in the absence of added protein. Further, correlation is demonstrated between the ability of these agents to inhibit NQO1 and computed binding affinity. We have solved the crystal structure of NQO1 complexed to a hybrid compound and find good agreement with the in silico model. For both MIA PaCa-2 pancreatic tumor cells and HCT116 colon cancer cells, dicoumarol shows the greatest toxicity of all compounds. Thus, we provide a computational, synthetic, and biological platform to generate competitive NQO1 inhibitors with superior pharmacological properties to dicoumarol. This will allow a more definitive study of NQO1 activity in cells, in particular, its drug activating/detoxifying properties and ability to modulate oncoprotein stability.

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The enzyme NAD(P)H quinone oxidoreductase (NQO1) can function both as a detoxifying enzyme as well as chaperone protein. The latter property has been extensively characterized by the use of dicoumarol which inhibits the chaperone properties of NQO1 in cells. However, the use of this compound is compromised by its multiple "off-target" effects. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of NQO1 in cells have been identified (Nolan et al., Biochem Pharmacol 2010;80:977-81). The purpose of the work reported here is to evaluate the off-target effects of these compounds when compared to dicoumarol. A range of these substituted coumarins are identified that are significantly less toxic than dicoumarol in a panel of nine cell lines. Further a number of the compounds generate much less intracellular superoxide, and many of them also show a reduced ability to induce apoptosis when compared to dicoumarol. None of these effects correlate with the ability of the compounds to inhibit the enzymatic activity of NQO1 in cells. In conclusion, potent inhibitors of NQO1 have been identified that will be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues.

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Imidazoacridin-6-ones are shown to be potent nanomolar inhibitors of the enzyme NQO2. By use of computational molecular modeling, a reliable QSAR was established, relating inhibitory potency with calculated binding affinity. Further, crystal structures of NQO2 containing two of the imidazoacridin-6-ones have been solved. To generate compounds with reduced off-target (DNA binding) effects, an N-oxide moiety was introduced into the tertiary aminoalkyl side chain of the imidazoacridin-6-ones. This resulted in substantially less toxicity in a panel of eight cancer cell lines, decreased protein binding, and reduced DNA binding and nuclear accumulation. Finally, one of the N-oxides showed potent ability to inhibit the enzymatic function of NQO2 in cells, and therefore, it may be useful as a pharmacological probe to study the properties of the enzyme in vitro and in vivo.

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ABSTRACT

Both impulsivity and stress are risk factors for substance abuse, but it is not clear how these two processes interact to alter susceptibility for the disorder. The aim of this project was to examine the pharmacology of a stress-impulsivity interaction in rats. To do so, we tested the effects of yohimbine on impulsive action and then assessed whether behavioural changes could be reduced by antagonists at different receptor subtypes. Male Long-Evans rats were injected with various doses of yohimbine (0-5.0 mg/kg) before testing in the response-inhibition task. In subsequent experiments, yohimbine (2.5 mg/kg) was injected following pretreatment with the following receptor antagonists: corticotropin-releasing factor receptor 1, antalarmin (0-20 mg/kg); glucocorticoid, mifepristone (0-30 mg/kg); noradrenergic (NA) α1, prazosin (0-2 mg/kg); NA α2, guanfacine (0-0.5 mg/kg); NA β2, propranolol (0.5-2.0 mg/kg); dopamine D1/5, SCH 39166 (0-0.0625 mg/kg); μ opioid, naloxone (0-2 mg/kg); or 5-HT2A, M100907 (0.005-0.05 mg/kg). In all experiments, impulsive action was measured as increased premature responding. Yohimbine dose dependently increased impulsive action, but the effect was not reversed by antagonist pretreatment. None of the drugs altered any other behavioural measure. We conclude that stress-impulsivity interactions are likely mediated by a synergy of multiple neurotransmitter systems.