Fluorescence resonance energy transfer substrates for determining cathepsin B pH specificity

Cathepsin B is a cysteine proteinase of the papain family that in malignant tumors is present both in lysosomes and in the pericellular space. At the acidic pH of lysosomes (4.5-5.5), it has mainly peptidyl-dipeptidase and carboxy-peptidase activity, its active site being partially occluded by a flexible loop; above pH 5.5 this loop is displaced and the enzyme behaves mainly as an endopeptidase having a pH optimum around 7.4. In the present study, we describe the preparation and the use of fluorescence resonance energy transfer (FRET) peptides to search for substrates selectively cleaved by cathepsin B at different pHs. Each peptide contained a highly fluorescent 2-(N-methylamino)benzoyl (Nma) group linked to the amino group of the Nterminal Orn residue. This group is efficiently quenched by a 2,4- dinitrophenyl (Dnp) group linked to the side-chain of a Lys residue. The development of substrates cleaved by the enzyme at neutral pH can provide useful information for the design of peptide pro-drugs releasing anticancer drugs in the pericellular space of tumor cells.