Significance of Specific Oxidoreductases in the Design of Hypoxia-Activated Prodrugs and Fluorescent Turn off–on Probes for Hypoxia Imaging

SIMPLE SUMMARY: Hypoxia-activated prodrugs (HAPs), selectively reduced by specific oxidoreductases under hypoxic conditions, form cytotoxic agents damaging the local cancer cells. On the basis of the reported clinical data concerning several HAPs, one can draw conclusions regarding their preclinical attractiveness and, regrettably, the low efficacy of Phase III clinical trials. Clinical failure may be explained, inter alia, by the lack of screening of patients on the basis of tumor hypoxia and low availability of specific oxidoreductases involved in HAP activation. There is surprisingly little information on the quantification of these enzymes in cells or tissues, compared to the advanced research associated with the use of HAPs. Our knowledge about the expression and activity of these enzymes in various cancer cell lines under hypoxic conditions is inadequate. Only in a few cases were researchers able to demonstrate the differences in the expression or activity of selected oxidoreductases, depending on the oxygen concentration. Additionally, it was cell line dependent. More systematic studies are required. The optical probes, based on turning on the fluorescence emission upon irreversible reduction catalyzed by the overexpressed oxidoreductases, can be helpful in this type of research. Ultimately, such sensors can estimate both the oxidoreductase activity and the degree of oxygenation in one step. To achieve this goal, their response must be correlated with the expression or activity of enzymes potentially involved in turning on their emissions, as determined by biochemical methods. In conclusion, the incorporation of biomarkers to identify hypoxia is a prerequisite for successful HAP therapies. However, it is equally important to assess the level of specific oxidoreductases required for their activation. ABSTRACT: Hypoxia is one of the hallmarks of the tumor microenvironment and can be used in the design of targeted therapies. Cellular adaptation to hypoxic stress is regulated by hypoxia-inducible factor 1 (HIF-1). Hypoxia is responsible for the modification of cellular metabolism that can result in the development of more aggressive tumor phenotypes. Reduced oxygen concentration in hypoxic tumor cells leads to an increase in oxidoreductase activity that, in turn, leads to the activation of hypoxia-activated prodrugs (HAPs). The same conditions can convert a non-fluorescent compound into a fluorescent one (fluorescent turn off–on probes), and such probes can be designed to specifically image hypoxic cancer cells. This review focuses on the current knowledge about the expression and activity of oxidoreductases, which are relevant in the activation of HAPs and fluorescent imaging probes. The current clinical status of HAPs, their limitations, and ways to improve their efficacy are briefly discussed. The fluorescence probes triggered by reduction with specific oxidoreductase are briefly presented, with particular emphasis placed on those for which the correlation between the signal and enzyme expression determined with biochemical methods is achievable.