Abstract 3105: Probing cytochrome P450 activity with benzofuran-based duocarmycins in a panel of head and neck cancer cell lines and CYP1A1, 1B1 and CYP2W1 transfected cell lines

Abstract

Abstract Background: The cytochrome P450 (CYP) isoforms CYP1A1, 1B1 and 2W1 are highly expressed in tumour tissue and surrounding stroma compared to nearby normal tissue, which provides an opportunity for development of selective cancer therapeutics as previously reported by us.1-2 However, no reagent is available to interrogate CYP2W1 and hence the purpose of this study was to evaluate a series of novel benzofuran-based duocarmycins as chemical probes to study the functional activity of CYP2W1. Materials and methods: Standard materials and methods can be found in the references below. Briefly, these include route to synthesis of duocarmycin bioprecursors, use of recombinant CYP bactosomes with LC/MS for metabolite identification, and cell culture MTT proliferation assay. CYP expression was measured by RT-PCR and western blot. Results: A panel of head and neck cancer (HNC) cell lines (SCC4, FaDu, Detroit-562, A-253, OSC19, UT-SCC5, UT-SCC10, UT-SCC14) were analysed for the expression of CYP1A1, CYP1B1 and CYP2W1. Significant expression (5-10 fold) was shown at the gene level while protein expression experiments are currently underway and will be reported at the meeting. Furthermore, CYP2W1 functional activity was probed with a novel series of benzofuran-based duocarmycin analogues. The compounds were incubated with recombinant CYP bactosomes or HEK2932W1-transfected cells and their oxidative metabolism was studied using LCMS. CYP2W1 was shown to metabolise one chemical probe, ICT2726, to a unique metabolite not observed for any other CYP isoform. The compounds were investigated for antiproliferative activity in a panel of cell lines (CHO, CHO1A1, RT112, HEK293, HEK2932W1, Cal27, Cal271B1, Cal33, Cal331B1). Obtained IC50 values > 1 μM, which for these ultrapotent (picomolar) duocarmycins, indicated no CYP-bioactivation, consistent with no active duocarmycin metabolite identified from the LCMS studies. Conclusion: Our findings support the use of non-toxic ICT2726 as a chemical tool that can be employed in vitro to probe CYP2W1 functional activity. [1] Sheldrake et al. Re-engineering of the duocarmycin structural architecture enables bioprecursor development targeting CYP1A1 and CYP2W1 for biological activity. J Med Chem. 2013, 56, 6273-7. [2] Travica et al. Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins. Clin Cancer Res. 2013, 19, 2952-61. Citation Format: Daniela Presa, Paul M. Loadman, Giuliano Nigro, Helen M. Sheldrake, Mark Sutherland, Steven D. Shnyder, Valerie Le Morvan, Jacques Robert, Magnus Ingelman-Sundberg, Laurence H. Patterson, Klaus Pors. Probing cytochrome P450 activity with benzofuran-based duocarmycins in a panel of head and neck cancer cell lines and CYP1A1, 1B1 and CYP2W1 transfected cell lines. [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 3105.