A New Fluorogenic Substrate for Granzyme B Based on Fluorescence Resonance Energy Transfer

The serine protease Granzyme B (GzmB) is a potent inducer of apoptosis in target cells when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells, representing one of the two dominant mechanisms by which T cells mediate cancer cell death. Considering the GzmB´s preference for cleaving after aspartic acid, several substrates containing IEPD and IETD sequences coupled to chromogenic or fluorescent leaving groups have been successfully used for GzmB detection. These two sequences, however, lack specificity as they can also be cleaved by some caspases. Also, the use of probes based on fluorescence resonance energy transfer (FRET) principle can have significant advantages over these singly-labelled probes.

Considering our interest in using FRET-based techniques to monitor GzmB activity in a controlled release system of immunostimulating drugs applied in the treatment of colorectal cancer, we reported herein the synthesis and characterization of a new fluorogenic GzmB substrate. For this, our substrate design was based on the FRET principle using 5-(2´-aminoethyl)aminonaphthalene sulfonic acid (Edans) and 4-[[4´-(N,N-dimethylamino)phenyl]diazenyl]benzoic acid (Dabcyl) as the energy transfer pair, linked to a specific sequence for GzmB (AAD), with an additional amino acid as anchoring point (K). The tetrapeptide was obtained by microwave assisted solid phase peptide synthesis and coupled to Dabcyl and Edans at its N- and C-termini, respectively. The obtained probe was purified by semi-preparative HPLC and characterized by NMR, UV/Vis absorption and fluorescence spectroscopy.