Design of a New Peptide Substrate Probe of the Putative Biomarker Legumain with Potential Application in Prostate Cancer
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Design of a New Peptide Substrate Probe of the Putative Biomarker Legumain with Potential Application in Prostate Cancer Diagnosis Ex Vivo Sunil Mathur1,4 · Agnes Turnbull1 · Iolia Akaev2,3 · Craig Stevens1 · Neerja Agrawal2 · Mridula Chopra3 · David Mincher1 Accepted: 9 December 2019 © The Author(s) 2019
Abstract The lysosomal endoprotease legumain (asparaginyl endoprotease) has been proposed as a putative biomarker in prostate tumours, in which the enzyme is markedly overexpressed. Overexpression, coupled with highly selective specificity for cleavage of substrates at the C-terminus of asparagine (Asn) residues, make legumain an attractive biochemical target for potential diagnosis, prognosis and treatment. We report the design, synthesis, characterisation and preliminary evaluation of a new rhodamine-B (Rho-B)-labelled legumain peptide substrate probe 5 [Rho-Pro-Ala-Asn-PEG-AQ(4-OH)] and its selective targeting to lysosomes in PC3 prostate cancer cells. Probe 5 was efficiently activated by recombinant human legumain to afford the high quantum yield reporter fluorophore tripeptide 4b (Rho-Pro-Ala-Asn-OH) with concomitant release of intense fluorescence. Furthermore, probe 5 was activated upon incubation with homogenates derived from fresh-frozen tissue material of prostatectomy specimens. Probe 5 represents a new viable biochemical tool for probing the activity of legumain with the potential to be used in ex vivo diagnostics in the cancer pathology laboratory. Keywords Prostate cancer · Biomarker · Legumain · Diagnosis · Synthesis
Introduction Prostate cancer (Pca) is one of the most prevalent cancers and its incidence is increasing in Western society (Siegel et al. 2016). The early diagnosis of pre-invasive and Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10989-019-09994-1) contains supplementary material, which is available to authorized users. * David Mincher [email protected] 1
School of Applied Sciences, Edinburgh Napier University, Edinburgh EH11 4BN, UK
2
Histopathology Laboratory, Portsmouth Hospitals NHS Trust, Pathology Centre, Histopathology, Queen Alexandra Hospital, Southwick Hill Road, Portsmouth PO6 3LY, UK
3
IBBS, School of Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michaels Building, White Swan Road, Portsmouth PO1 2DT, UK
4
Present Address: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK
metastatic tumour cells in patients is pivotal for the judicious choice of treatment and success of therapies and for the improvement of survival rates. Early diagnosis of Pca is usually reliant on techniques that include digital rectal examination, magnetic resonance imaging and transrectal ultrasound-guided biopsy in conjunction with screening for serum levels of prostate specific antigen (PSA) which remains the principal biomarker for Pca (Zhang et al. 2017). A challenge across the spectrum of cancers is to identify and incorporate int
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