Carbon Monoxide Prodrugs for Targeted Cancer Therapy

Abstract

Carbon monoxide (CO) is endogenously produced in the body through the breakdown of haem by haem oxygenase enzymes. Since this discovery, CO has been recognised as a gasotransmitter alongside nitric oxide and hydrogen sulfide. Beyond established physiological roles, CO has shown therapeutic potential in many pathological and clinical conditions, including inflammation and cancer. However, challenges in targeted and controlled delivery have limited its clinical application, prompting the development of novel CO-releasing prodrugs. This thesis presents the design and development of radical-activated, metal-free CO-prodrugs. The prodrugs are designed to address delivery limitations and avoid metal-associated toxicity. These tertiary aldehyde-based prodrugs are stable under physiological conditions and, upon activation by a radical trigger, release CO, 2-ethyl-1-butene, and a non-toxic thiol carrier. CO was released in response to both exogenous and endogenous stimuli. The release of other products was detected by both MS and NMR. Quantum mechanical calculations also supported the feasibility of the hypothesised mechanism under physiological conditions. The prodrugs demonstrated CO-like therapeutic effects both in vitro and in vivo. Attempts were made to mask the aldehyde functional group or improve physiochemical properties through structure–activity relationship studies, but no superior alternatives were identified. The stability of such CO-prodrugs allowed for their incorporation into synthetic peptides via solid-phase peptide synthesis and subsequent site-specific bioconjugation to therapeutic antibodies. Trastuzumab–CO-prodrug conjugates were synthesised with CO-to-antibody ratios of up to an average of 23 and demonstrated tumour-specific CO release in HER2-overexpressing cells. The platform was subsequently adapted to anti-PD-L1 miniprotein conjugates, allowing selective CO delivery to PD-L1–expressing cancer cells. To the best of our knowledge, this represents the first example of biomarker-targeted, cancer-specific CO release. As a complementary project, the previously developed bioconjugation and CO-detection strategies were applied to a cathepsin B–responsive, iron-core CO-prodrug, providing a second example of targeted delivery of CO based on cancer-associated biomarkers. These findings open new avenues for investigating the therapeutic effects of CO. We anticipate that our CO-prodrug strategy will be broadly applicable to a wide range of synthetic peptide- and protein-based therapeutics.