Hypoxia (conditions of reduced oxygen) occurs in a wide variety of biological environments, including solid tumours and bacterial biofilms, and has negative implications for cancer patient prognosis. Hypoxia-activated prodrugs offer the ability to deliver compounds selectively to regions of hypoxia, through attachment of bioreductive groups, (e.g. 4-nitrobenzyl moiety). Oxygen-dependent nitroreductase enzymes catalyse reduction of the nitro group to form a radical nitro anion, which is re-oxidised back to the nitro group in the presence of oxygen. Under hypoxic conditions, this futile REDOX cycling cannot occur. The nitro anion radical is further reduced to form a hydroxylamine or amine; subsequent fragmentation releases the drug. This work combines the concept of hypoxia activation with an IPTG inducible gene expression system. Under uninduced conditions, the lac repressor (LacI) binds to the DNA, preventing RNA polymerase from initiating transcription. Following addition, IPTG binds to LacI, which dissociates from the DNA, allowing transcription and subsequent translation of the downstream gene. We hypothesised that protecting IPTG with a bioreductive group would inhibit binding of IPTG to LacI. Only under hypoxic conditions will reduction of the bioreductive and release of IPTG occur, resulting in hypoxia-selective gene expression (Figure 1A). A range of 4-nitrobenzyl derivatives of IPTG were synthesised and analysed for their ability to undergo reduction and fragmentation to release IPTG. Using GFP as a reporter gene, these were tested in BL21 (DE3) E. coli. Through fluorescence and western blot analysis, it was shown that 1 successfully induces expression of GFP selectively in hypoxia (Figure 1B). This compound is being used to investigate translating the technology in a range of settings, including mammalian and plant cells.

Hypoxia activated small molecule induced gene expression

256th National Meeting (2018)

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