Colon Cancer–Specific Cytochrome P450 2W1 Converts Duocarmycin Analogues into Potent Tumor Cytotoxins

Abstract

Purpose: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents.Experimental Design: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts.Results: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue.Conclusion: Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1.

Probing cytochrome P450-mediated activation with a truncated azinomycin analogue

“…Certain isoforms, notably CYP1A1, 1B1 and 3A4 are identified as highly expressed in certain tumours 10 indicating the value of truncated azinomycin analogues as potential bioprecursors with chemotherapeutic efficacy. We have demonstrated proof of principle with bioprecursors of the duocarmycin natural products targeting CYP1A1 19,20 and 2W1 14,21 enzymes. Uniquely, we have demonstrated that CYP selective bioactivation in vivo can be obtained with no overt toxicity to normal tissues including the liver.…”

“…Uniquely, we have demonstrated that CYP selective bioactivation in vivo can be obtained with no overt toxicity to normal tissues including the liver. 13,14 Further development of such a bioprecursor approach could provide a strategy to exploit the azinomycins for therapeutic use. While CYP1A1 and 1B1 provide selective cancer drug targets, the hepatic CYP3A4 enzyme could be exploited for liver specific treatment of hepatitis B, hepatitis C and hepatocellular carcinoma 22 or with the reemergence of the oncolytic virus, a 'suicide' GDEPT approach also exists as a therapeutic strategy.…”

“…We developed AQ4N, a Phase II clinical trial candidate, to undergo bioreductive activation by CYP1A1 and 3A4 selectively in hypoxic tumours. 11,12 More recently we demonstrated that bioprecursors of the duocarmycins can be activated selectively to ultrapotent anti-proliferative metabolites by tumours expressing CYP1A1 13 and 2W1 14 and we are proposing this approach to address the failure of the duocarmycins in the clinic due to unacceptable normal tissue toxicity. Similarly, despite the promise of the azinomycins as cancer chemotherapeutics, they have thus far failed to progress clinically.…”

The CYP2W1 enzyme: regulation, properties and activation of prodrugs

“…It also appeared that the cytotoxic metabolite of chloromethylindolines can be transferred from one tumor cell to others in close proximity, thus indicating a bystander propagating effect (Sheldrake et al, 2013;Travica et al, 2013).…”

Rational Development of Novel Activity Probes for the Analysis of Human Cytochromes P450

“…Given their importance,p articularly in xenobiotic and drugm etabolism, [1] ag reat deal of research has been conducted into their roles, the identification of their sequences, and their catalytic mechanisms. [2][3][4] Whilst an umber of CYPs, particularly human liver CYPs and extrahepatic CYPs (i.e.,C YP1A1, CYP1B1, and CYP2W1)h ave been the subjecto f intense investigation, [5][6][7] there is stillm uch to be learnt from these mixed-function oxidases. This reflects the difficulties associated with studyingt hese enzymes,because:1 )they are encoded by large gene families, and their functions cannotb e predicted fromt heir gene sequence;2 )they are difficult to assay,i solate and purify, so classical biochemical methods are often ineffective in identifying enzymes of interest;3 )these proteins are membrane bound and often depend on co-enzymes and cofactors, making them difficult to express as functional enzymesi nc ellular systems; and 4) polymorphismsa nd epigenetic regulationa lter their expression levels and functional activity.T herefore, new approaches to identifying, evaluating, and quantifyingf unctionally active CYPs are of utmost importance.…”