Fluorescent probes for proteolysis: Tools for drug discovery

Abstract

Our body contains many different protease and proteolytic systems that are involved in the recycling of proteins into amino acids, and also in a multitude of regulatory events inside and outside cells. Proteases are prominent drug targets because of their well-defined chemistry and their implication in a large number of diseases, such as cancer, neurodegeneration, arteriosclerosis, inflammation and infection.Fluorescent reporter substrates can be used to directly probe the activities of proteases in their natural environment — that is, in cells and organisms. Conceptually different strategies have been used for this purpose depending on the location and the nature of the protease of interest.Fluorescent reporters for the ubiquitin–proteasome system have been generated by linking constitutively active degradation signals to green fluorescent protein (GFP). These GFP-based substrates can be used for functional analysis of the ubiquitin–proteasome system in cells and transgenic animals.A collection of different proteases is involved in degradation of small peptide fragments. This process can be followed in real time in living cells by confocal laser scanning microscopy after microinjection of internally quenched peptide substrates.Extracellular and lysosomal proteases have the advantage that they are accessible for membrane-impermeable reporter substrates. Near-infrared fluorescence (NIRF) substrates are quenched fluorescent peptides that, because of their near-infrared excitation, can be readily detected in living animals and used for in vivo monitoring of, for example, lysosomal cathepsins or surface matrix metalloproteinases.By combining specific pairs of fluorescent proteins (GFP and its variants with shifted excitation and emission spectra) in fusion proteins, fluorescence energy transfer (FRET) reporter substrates have been generated for initiator and effector caspases.A fluorescent intracellular reporter for human immunodeficiency virus (HIV-1) protease activity was constructed by fusing a protease precursor protein composed of HIV-1 protease and GFP. Cells will only survive and emit fluorescence when the toxic protease activity is sufficiently blocked by drugs.The diffusion rate of the endoplasmic reticulum-resident peptide transporter complex TAP correlates with activity and thus cytosolic peptide levels. By measuring the diffusion of TAP–GFP fusions with fluorescence recovery after photobleaching (FRAP), the kinetics of peptide generation can be followed in living cells.