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Design, synthesis and evaluation of novel endoprotease-activated prodrugs and fluorogenic probes with theranostic applications in chemotherapy

Ding, Yao


Yao Ding


Proteolytic degradation of the extracellular matrix (ECM) by overexpressed endoproteases, notably the matrix metalloproteinase MMP-9 and legumain (human asparaginyl endopeptidase), contributes to the invasive and migratory phenotype of cancer cells and promotes metastases. These proteases represent valid biological targets in cancer for diagnostic and therapeutic purposes.
In this research programme, novel MMP-substrate oligopeptide prodrugs of potent anticancer agents have been rationally designed for activation selectively in the tumour microenvironment. Prodrugs of experimental colchicine-based cytotoxic and vascular disrupting agents, experimental anthraquinone-based agents or clinically-active drugs, including epirubicin, have been synthesised by both solution- and solid-phase peptide methods, and characterised. Results of the preliminary cytotoxic and DNA-binding properties of examples of active agents are reported.
Furthermore, the approach has been extended to legumain, which is overexpressed in colon, breast and ovarian cancers and is capable of extracellular proteolytic activity. A series of novel substrates of legumain has been designed and characterised by high resolution mass spectrometry. The new peptide substrates have been developed as fluorogenic molecular probes of legumain for diagnostic applications in the early detection of cancer. The novel substrates exploit the proteolytic activity of legumain to cleave uniquely at the C-terminus of asparagine residues. The new peptide substrates, exemplified by prototype fluorogenic probe TL11 (FAM-Pro-Ala-Asn-Leu-PEG-AQ) are efficient FRET substrates in which fluorescein-based (donor) fluorescence is fully quenched by an aminoanthraquinone residue (acceptor). Proof of principle has been demonstrated by use of recombinant human legumain which cleaves the substrate library efficiently as shown by fluorescence spectroscopic methods. The tetrapeptide sequence pro-X-asn↓-leu was shown to be sensitive when X= ala, ser or thr (wherein ↓indicates the legumain cleavage ‘hotspot’). The approach was also shown to be extendable to a therapeutic prodrug approach with the potential to selectively deliver potent agents to the tumour microenvironment.

Thesis Type Thesis
Deposit Date Feb 12, 2015
Peer Reviewed Not Peer Reviewed
Keywords Cancer; proteases; biological targets; extracellular matrix (ECM); Proteolytic degradation; metalloproteinase MMP-9; legumain (human asparaginyl endopeptidase);
Public URL
Contract Date Feb 12, 2015
Award Date 2014-01


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