A Coumarin-Porphyrin FRET Break-Apart Probe for Heme Oxygenase-1

Heme oxygenase-1 (HO-1) is a critical determinant of vascular homeostasis, health and disease. HO-1 deficiency causes death from overwhelming vascular disease in childhood. HO-1 degrades heme, which is potentially toxic due to oxidative stress. HO-1 is induced by tissue hemorrhage, which it helps resorb. HO-1 is induced in hemorrhages in atherosclerotic lesions (intraplaque hemorrhage (IPH), in intracranial hemorrhage in stroke, and in ruptured atherosclerotic abdominal aneurysm. However, the measurement of HO-1 activity is cumbersome and restricted to prolonged assays in cell lysates. A simple, easily used real-time activity probe would speed up research and may be an extremely useful diagnostic that would allow early and sensitive detection of intraplaque hemorrhage.

A partnership between Joe Boyle in National Heart and Lung Institute and Nick Long in the Molecular Sciences Research Hub – particularly postdocs Ed Walter and Ying Ge - has designed, synthesised, and characterised entirely an new fluorescent probe for HO-1. This molecule glows blue in the presence of ultraviolet light when Heme Oxygenase has acted on it. This was the first application of the Fluorescence Energy Transfer (FRET) breakapart approach to a small molecule metabolite (rather than an oligopeptide or oligonucleotide). In this, HO-1 removes a coumarin from precisely one position, preventing transfer of the fluorescence excitation from the coumarin to the porphyrin. Thereby, when the probe sees the enzyme, blue fluorescence emission results.

Analysis of HO-1 activity from cells including heme-stimulated human blood-derived macrophages found a substantial increase in blue fluorescence emission following incubation with the probe. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC−MS, showing predicted products of the probe that following processing by HO-1. Importantly, analysis of a control inactive analogues showed that close structural analogues of heme are required to maintain HO-1 activity. This work has triggered the design of new probes for HO-1 activity, that we will develop and use in live applications in cells and in vivo models.

This is a genuinely equal and mutual partnership that has been facilitated by the move of the Department of Chemistry to the Molecular Sciences Research Hub (MSRH) in the new White City Campus, and by the numerous collaborative contacts fostered between the Faculties at Imperial College London. The work was inconceivable except as an inter-Faculty and interdisciplinary venture.

Link to publication: A Coumarin-Porphyrin FRET Break-Apart Probe for Heme Oxygenase-1 - PubMed (nih.gov)