Solid tumours are often poorly oxygenated, which confers resistance to standard treatment modalities. Targeting hypoxic tumours requires compounds, such as
nitroimidazoles (NIs), equipped with the ability to reach and become activated within diffusion limited tumour niches. NIs become selectively entrapped in hypoxic cells through bioreductive activation, and have shown promise as
hypoxia directed
therapeutics. However, little is known about their mechanism of action, hindering the broader clinical usage of NIs. Iodoazomycin arabinofuranoside (
IAZA) and fluoroazomycin arabinofuranoside (FAZA) are clinically validated 2-NI hypoxic radiotracers with excellent tumour uptake properties. Hypoxic
cancer cells have also shown preferential susceptibility to
IAZA and FAZA treatment, making them ideal candidates for an in-depth study in a therapeutic setting. Using a
head and neck cancer model, we show that hypoxic cells display higher sensitivity to
IAZA and FAZA, where the drugs alter cell morphology, compromise DNA replication, slow down cell cycle progression and induce replication stress, ultimately leading to cytostasis. Effects of
IAZA and FAZA on target cellular macromolecules (
DNA,
proteins and
glutathione) were characterized to uncover potential mechanism(s) of action. Covalent binding of these NIs was only observed to cellular
proteins, but not to
DNA, under
hypoxia. While
protein levels remained unaffected, catalytic activities of NI target
proteins, such as the glycolytic
enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the detoxification
enzyme glutathione S-transferase (GST) were significantly curtailed in response to
drug treatment under
hypoxia. Intraperitoneal administration of
IAZA was well-tolerated in mice and produced early (but transient) growth inhibition of subcutaneous mouse tumours.