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.
Highlights
Prostate cancer (Pca) is one of the most prevalent cancers and its incidence is increasing in Western society (Siegel et al 2016)
A fluorescence resonance energy transfer (FRET) system was designed to provide for continuous reaction monitoring and rapid determination of enzyme activity and that could be envisaged as a potential simple tool for use in the cancer pathology laboratory, requiring inexpensive instrumentation; notably, to detect legumain biomarker activity in prostate tissue samples
The design of probe 5 [Rho-Pro-Ala-Asn-PEG-AQ(4-OH)] (Fig. 1) as a new FRET peptide substrate of legumain, incorporating a rhodamine B derivative modified at the 2 ́-carboxylic acid group was selected on the basis of its favourable photostability, high relative fluorescence quantum yield, long emission wavelength, low cost and ease of access by chemical synthesis
Summary
Prostate cancer (Pca) is one of the most prevalent cancers and its incidence is increasing in Western society (Siegel et al 2016). 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). The lack of tumour specificity of PSA can lead to misdiagnosis and over diagnosis of low-risk Pca (Dijkstra et al 2012). Such over diagnosis is a consequence in the so-called ‘grey area’ of PSA levels between 4.0 and 10.0 ng/mL. Potential alternative biomarkers have been proposed and are the subject of recent review (Saini 2016), to
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