Edinburgh Napier University

  Despite great advancement in the diagnosis and treatment of many cancers, mortality rates remain high and the incidence of some forms of cancer is increasing.

Early diagnosis is recognised as an important factor to increase survival but there is currently a lack of reliable biomarkers of disease.

The focus of this research is the development of a new molecular probe for the putative cancer biomarker legumain (asparaginyl endoprotease) which is overexpressed in a wide variety of cancers and has been proposed as a viable biomarker of disease progression.

A novel FRET peptide substrate AQ(4-OH)-β-Ala-Pro-Ala-Asn-γ-Abu-piperazine-Rho (DR21) for legumain was designed with a peptide sequence containing a critical asparagine residue (cleavage site) at the P1 position for recognition and cleavage by the target enzyme. Uniquely, the N-terminus was capped by an aminoanthraquinone quencher (acceptor) group and a rhodamine B-derived fluorophore was incorporated at the C-terminus. DR21 was characterised by high resolution mass spectrometry and by NMR spectroscopy and efficient quenching of rhodamine fluorescence was demonstrated.

Proof of principle was demonstrated by fluorescence spectroscopy. DR21 was activated upon incubation with recombinant human legumain with concomitant release of intense rhodamine fluorescence consistent with legumain-induced activation of the fluorogenic probe. The ability to release a hydrophobic derivative of rhodamine has potential advantages over existing fluorogenic substrates due to possessing structural features to allow entry to cells to aid imaging for tumours that express legumain.