CYP450 Enzymes Effect Oxygen-Dependent Reduction of Azide-Based Fluorogenic Dyes.

Azide-containing compounds have broad utility in organic synthesis and chemical biology. Their use as powerful tools for the labeling of biological systems in vitro has enabled insights into complex cellular functions. To date, fluorogenic azide-containing compounds have primarily been employed in the context of click chemistry and as sensitive functionalities for hydrogen sulfide detection. Here, we report an alternative use of this functionality: as fluorogenic probes for the detection of depleted oxygen levels (hypoxia). Oxygen is imperative to all life forms, and probes that enable quantification of oxygen tension are of high utility in many areas of biology. Here we demonstrate the ability of an azide-based dye to image hypoxia in a range of human cancer cell lines. We have found that cytochrome P450 enzymes are able to reduce these probes in an oxygen-dependent manner, while hydrogen sulfide does not play an important role in their reduction. These data indicate that the azide group is a new bioreductive functionality that can be employed in prodrugs and dyes. We have uncovered a novel mechanism for the cellular reduction of azides, which has implications for the use of click chemistry in hypoxia.

Design, synthesis and evaluation of molecularly targeted hypoxia-activated prodrugs.

Regions of insufficient oxygen supply-hypoxia-occur in diverse contexts across biology in both healthy and diseased organisms. The difference in the chemical environment between a hypoxic biological system and one with normal oxygen levels provides an opportunity for targeting compound delivery to hypoxic regions by using bioreductive prodrugs. Here we detail a protocol for the efficient synthesis of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol, which is a key intermediate that can be converted into a range of 1-methyl-2-nitro-1H-imidazole-based precursors of bioreductive prodrugs. We outline methods for attaching the bioreductive group to a range of functionalities, and we discuss the strategy for positioning of the group on the biologically active parent compound. We have used two parent checkpoint kinase 1 (Chk1) inhibitors to exemplify the protocol. The PROCEDURE also describes a suite of reduction assays, of increasing biological relevance, to validate the bioreductive prodrug. These assays are applied to an exemplar compound, CH-01, which is a bioreductive Chk1 inhibitor. This protocol has broad applications to the development of hypoxia-targeted compounds.

CH-01 is a hypoxia-activated prodrug that sensitizes cells to hypoxia/reoxygenation through inhibition of Chk1 and Aurora A.

The increased resistance of hypoxic cells to all forms of cancer therapy presents a major barrier to the successful treatment of most solid tumors. Inhibition of the essential kinase Checkpoint kinase 1 (Chk1) has been described as a promising cancer therapy for tumors with high levels of hypoxia-induced replication stress. However, as inhibition of Chk1 affects normal replication and induces DNA damage, these agents also have the potential to induce genomic instability and contribute to tumorigenesis. To overcome this problem, we have developed a bioreductive prodrug, which functions as a Chk1/Aurora A inhibitor specifically in hypoxic conditions. To achieve this activity, a key functionality on the Chk1 inhibitor (CH-01) is masked by a bioreductive group, rendering the compound inactive as a Chk1/Aurora A inhibitor. Reduction of the bioreductive group nitro moiety, under hypoxic conditions, reveals an electron-donating substituent that leads to fragmentation of the molecule, affording the active inhibitor. Most importantly, we show a significant loss of viability in cancer cell lines exposed to hypoxia in the presence of CH-01. This novel approach targets the most aggressive and therapy-resistant tumor fraction while protecting normal tissue from therapy-induced genomic instability.