Design of a New Peptide Substrate Probe of the Putative Biomarker Legumain with Potential Application in Prostate Cancer Diagnosis Ex Vivo

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.

Role of LGMN in tumor development and its progression and connection with the tumor microenvironment

“…The cysteine protease known as legumain is most commonly located in lysosomes, which are located in highly acidic ( Liu et al, 2003 ; Edgington et al, 2013 ; Mai et al, 2017 ; Mathur et al, 2020 ). Legumain up-regulation is associated with tumor invasion, proliferation, and angiogenesis ( Lin et al, 2020a ).…”

Peptide probes for proteases – innovations and applications for monitoring proteolytic activity

“…These include a variety of combinatorial approaches (both solid 21,22 and solution based, 23 MS substrate profiling methods, 24,25 phage libraries 26 or substrate modification of known substrates, including direct screening on complex tumour tissues. 27 Many FRET peptide probes have been developed [28][29][30] and there are reviews covering this area so here we highlight some key and novel examples 14,19 with FRET based probes developed for legumain, 31 thrombin, 30 SARS-CoV protease, 32 Granzyme B 33 and Cathepsins, 34 among others.…”

Development of a Promising 18F-Radiotracer for PET Imaging Legumain Activity In Vivo

“…Legumain, a cysteine protease, mainly exists in the lysosome under acidic environment [ 6 , 7 , 8 , 9 ]. The up-regulation of legumain is highly associated with cancer invasion, metastasis and angiogenesis [ 10 ].…”