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Design and evaluation of novel fluorogenic probes and prodrugs in cancer.

Mather, Sunil


Sunil Mather


Despite major advances in the diagnosis and treatment of cancer, there remains a paucity of biomarkers for early detection (poor selectivity and specificity). Legumain [asparaginyl endopeptidase (AEP); EC] is a potential cancer biomarker and molecular target for imaging and therapy. Legumain is a lysosomal protease, active at acidic pH (4.0 - 6.5) with a remarkably restricted substrate specificity, uniquely cleaving an asparagine (Asn) at the P1 position, and is overexpressed in various solid tumours. A novel legumain-targeted first generation fluorogenic rhodamine-B based peptide substrate Rho-Pro-Ala-Asn~PEG-AQ (SM9) has been developed for diagnostic application in the early detection of tumours, which exploits the enzyme’s proteolytic specificity. The fluorogenic probe SM9 is an efficient FRET substrate, in which an aminoanthraquinone acts as a ‘black hole’ quencher of rhodamine fluorescence that is restored on incubation with recombinant (rh)-legumain. Importantly, confocal microscopy studies have revealed localization of SM9 in the lysosomes of PC3 prostate cancer cells. The design principles have been extended to a second generation orthogonally functionalised, legumain-activated dual fluorogenic gadolinium-based magnetic resonance imaging (MRI) contrast agent Rho-Pro-Ala-Asn~Lys-[DOTA-(Gd3+)]-PEG-AQ (SM32). Furthermore, towards the development of selective and targeted theranostic (therapeutic and diagnostic) anticancer agents, a novel third generation fluorogenic legumain substrate prodrug Rho-Pro-Ala-Asn~Propyl-Pip-Propyl-AQ (ALS5) that incorporates a cytotoxic, lysosomotropic anthracenedione ALS1, has been designed. Activation of prodrug ALS5 by rh-legumain in vitro directly afforded the cleavage product and active drug ALS1. Confocal microscopy studies have shown that ALS5 and active ALS1 (at 1 μM) were also localized in the lysosomes of PC-3 cells. Furthermore, ALS1 induced morphological changes and apoptosis in PC-3 cells, as measured by fluorescence microscopy, and staining with Annexin V and DAPI using flow cytometry. All novel legumain-activated oligopeptide substrates and intermediate compounds have been fully characterised by high resolution mass spectrometry and NMR spectroscopy. Selected compounds have been further characterised by HPLC. The molecular probes and prodrugs have the potential to be used as diagnostic tools to define the legumain expression in tumour biopsies and provide prognostic information of value in determining patient-focussed treatment options, with extension to therapeutic strategies to improve tumour targeting.

Thesis Type Thesis
Deposit Date Feb 6, 2018
Publicly Available Date Feb 6, 2018
Keywords Cancer, Prodrugs, Fluorogenic Probes, Design, Evaluation,
Public URL
Contract Date Feb 6, 2018
Award Date 2017-10


Design and evaluation of novel fluorogenic probes and prodrugs in cancer. (84.4 Mb)

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