Edinburgh Napier University

  Background: In spite of major advances in the diagnosis and treatment of cancer, there remains a paucity of biomarkers for early detection. Legumain is a potential cancer biomarker and a molecular target for imaging and drug targeting. Legumain is a lysosomal protease with a remarkably restricted specificity, as it cleaves only substrate sequences having an asparagine (Asn) at the P1 site. We have previously shown that a legumain substrate probe (SM9), Pro-Ala-Asn~PEG-AQ, is quenched until activated by proteolytic action of legumain. Design of first generation molecular probe SM9 has been extended to a second generation theranostic dual probe-prodrug SM20 (RhoPro-Ala-Asn~Lys-PEG-AQ) that serves as both a diagnostic and therapeutic (theranostic) tool for cancer.
Material and Methods: Solution and solid phase peptide methods have been used to create a novel rhodamine-labelled tetra peptide second generation theranostic dual probe-prodrug (SM20) of legumain, which was purified by chromatographic methods and characterised by high resolution mass spectrometry, together with all synthetic intermediates. Fluorescence spectroscopic methods were used to determine the efficiency of FRET in the prodrug (SM20). In vitro fluorimetric assay was developed with human
recombinant legumain at 370C in MES assay buffer (pH 5.0). The lipophilic character (Log D) of the SM20 was assessed by distribution coefficient measurements. Confocal microscopy studies were performed to investigate the cellular uptake and sub-cellular localization of both SM9 and SM20 and their legumain-mediated cleavage fragments in PC3 prostate cancer cells.
Results: The fluorogenic second generation prodrug (SM20) is an efficient FRET substrate and affords good restoration of fluorescence when incubated at 10mM with rh-legumain (40 ng) in legumain assay buffer (pH-5.0; lex 544 nm, lem 585 nm). Cleavage at the designed Asn~Lys (SM20) cleavage ‘hotspot’ was demonstrated at concentrations of legumain in the 5−40 ng range in in vitro metabolism experiments using recombinant legumain. The second generation theranostic prodrug (SM20) is structurally similar to the SM9 probe, with a different aminoanthraquinone quencher, which is lysosomotropic.
Measurement of the distribution coefficients have shown that SM20 is more lipophilic than the Lys-PEG-AQ cleavage product from which it is derived. Confocal substrate for sensitive and early detection of legumain and a smart therapeutic agent for cancer. Work is ongoing to optimize the dual probe-prodrug and to extend the studies in vivo.