Summary Tumor hypoxia is associated with therapy resistance and poor patient prognosis. Hypoxia-activated prodrugs, designed to selectively target hypoxic cells while sparing normal tissue, represent a promising treatment strategy. We report the pre-clinical efficacy of 1-methyl-2-nitroimidazole panobinostat (NI-Pano, CH-03), a novel bioreductive version of the clinically used lysine deacetylase inhibitor, panobinostat. NI-Pano was stable in normoxic (21% O 2 ) conditions and underwent NADPH-CYP-mediated enzymatic bioreduction to release panobinostat in hypoxia (<0.1% O 2 ). Treatment of cells grown in both 2D and 3D with NI-Pano increased acetylation of histone H3 at lysine 9, induced apoptosis, and decreased clonogenic survival. Importantly, NI-Pano exhibited growth delay effects as a single agent in tumor xenografts. Pharmacokinetic analysis confirmed the presence of sub-micromolar concentrations of panobinostat in hypoxic mouse xenografts, but not in circulating plasma or kidneys. Together, our pre-clinical results provide a strong mechanistic rationale for the clinical development of NI-Pano for selective targeting of hypoxic tumors.

Hypoxia is a feature of most solid tumours and predicts for poor prognosis. In radiobiological hypoxia (&lt;0.1% O2) cells become up to three times more resistant to radiation. The biological response to radiobiological hypoxia is one of few physiologically relevant stresses that activates both the unfolded protein and DNA damage responses (UPR and DDR). Links between these pathways have been identified in studies carried out in normoxia. Based in part on these previous studies and recent work from our laboratory, we hypothesised that the biological response to hypoxia likely includes overlap between the DDR and UPR. While inhibition of the DDR is a recognised strategy for improving radiation response, the possibility of achieving this through targeting the UPR has not been realised. We carried out a systematic review to identify links between the DDR and UPR, in human cell lines exposed to &lt;2% O2. Following PRISMA guidance, literature from January 2010 to October 2020 were retrieved via Ovid MEDLINE and evaluated. A total of 202 studies were included. LAMP3, ULK1, TRIB3, CHOP, NOXA, NORAD, SIAH1/2, DYRK2, HIPK2, CREB, NUPR1, JMJD2B, NRF2, GSK-3B, GADD45a, GADD45b, STAU1, C-SRC, HK2, CAV1, CypB, CLU, IGFBP-3 and SP1 were highlighted as potential links between the hypoxic DDR and UPR. Overall, we identified very few studies which demonstrate a molecular link between the DDR and UPR in hypoxia, however, it is clear that many of the molecules highlighted warrant further investigation under radiobiological hypoxia as these may include novel therapeutic targets to improve radiotherapy response.

Hypoxia (low oxygen levels) occurs in a range of biological contexts, including plants, bacterial biofilms, and solid tumors; it elicits responses from these biological systems that impact their survival. For example, conditions of low oxygen make treating tumors more difficult and have a negative impact on patient prognosis. Therefore, chemical probes that enable the study of biological hypoxia are valuable tools to increase the understanding of disease-related conditions that involve low oxygen levels, ultimately leading to improved diagnosis and treatment. While small-molecule hypoxia-sensing probes exist, the majority of these image only very severe hypoxia (<1% O2) and therefore do not give a full picture of heterogeneous biological hypoxia. Commonly used antibody-based imaging tools for hypoxia are less convenient than small molecules, as secondary detection steps involving immunostaining are required. Here, we report the synthesis, electrochemical properties, photophysical analysis, and biological validation of a range of indolequinone-based bioreductive fluorescent probes. We show that these compounds image different levels of hypoxia in 2D and 3D cell cultures. The resorufin-based probe 2 was activated in conditions of 4% O2 and lower, while the Me-Tokyo Green-based probe 4 was only activated in severe hypoxia0.5% O2 and less. Simultaneous application of these compounds in spheroids revealed that compound 2 images similar levels of hypoxia to pimonidazole, while compound 4 images more extreme hypoxia in a manner analogous to EF5. Compounds 2 and 4 are therefore useful tools to study hypoxia in a cellular setting and represent convenient alternatives to antibody-based imaging approaches.