Evaluating Novel 4-Aminoquinoline Inhibitors Of Nqo2 In Ovarian Cancer

Introduction: NRH quinone oxidoreductase 2 (NQO2) is one of the mammalian enzymes responsible for the two-electron reduction of quinones to hydroquinones. NQO2 can use a variety of dihydronicotinamide analogues as co-factors. In particular, the co-factor, EP0152R has been used in Phase I clinical trials to support evaluation of the NQO2-mediated reductive activation of CB1954. As well as being a prodrug-activating enzyme, there is evidence to show that NQO2 may be involved in cancer progression as a consequence of its ability to act as a “nanny” protein, alter cyclin D expression and modulate the activity of NF-κB. Thus, we have synthesized a series of novel 4-aminoquinolines to study their ability to inhibit NQO2 in ovarian cancer cells (OVCs). We then propose to compare the results of pharmacological inhibition with those obtained by genetic down-regulation of NQO2 level. Methods: NQO2 level in a panel of OVC cell lines was determined by using western blot analysis and Cytochrome C based spectrophotometric enzyme activity assay. Novel compounds have been synthesized and assessed for their inhibitory potency against recombinant human NQO2 (rhNQO2), using DCPIP spectrophotometric assay. MTT cytotoxicity assay was utilized for several purposes: Firstly, to determine the toxicity of known and novel NQO2 inhibitors in the SKOV3 cell line; and secondly, to evaluate the sensitivity of OVC cell lines to CB1954 in the presence of EP0152R, and subsequently measure the intracellular NQO2 inhibitory potency of novel compounds, by combining them with CB1954 and EP0152R. Results and conclusion: In the OVCs, NQO2 protein level and enzymatic activity showed an excellent correlation; however, expression and activity differed widely among the OVC cell lines. The NQO2 levels were highest in SKOV3 and lowest in the TOV112D cell lines. The sensitivity of OVCs to CB1954 was significantly increased when combined with EP0152R; supporting the notion that NQO2 mediates the toxicity of CB1954. This was confirmed when a strong correlation was observed between the cellular NQO2 activity and the responsiveness of the OVC cell lines to CB1954. Further, overexpressing NQO2 in TOV112D cells resulted in a significant enhancing of its responsiveness to treatment with CB1954. The novel 4-aminoquinolines inhibited the activity of recombinant NQO2 when used in the nano-molar range. The inherent cytotoxicity of the inhibitors (as measured by the IC50 following 24 and 96 hours exposure to SKOV3 cells) ranged between 1.7 to u003e100μM. There was no relationship between toxicity and the ability of the compounds to inhibit the activity of rhNQO2. However, some of these compounds showed functional activity as NQO2 inhibitors in cells; this was revealed by their ability to inhibit the toxicity of CB1954. Several compounds were able to do this at sub-μM, non-toxic concentrations and therefore they may be able to act as molecular probes for the (multi-) functional activity of NQO2 in cells. Citation Format: Balqis A. Ikhmais, Buthaina Hussein, Ian Stratford, Sally Freeman, Joaana Neill. Evaluating novel 4-aminoquinoline inhibitors of NQO2 in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3007.