Cellular proteases play a fundamental role in many cellular processes and are intimately involved in the development of many diseases, thus representing valuable targets for therapeutic intervention. Biochemical assays using peptide-conjugated substrates are widely used for characterising proteases.
Although these assays are convenient for compound screening, they are poorly informative about the complex and the dynamic changes taking place in live cells. Therefore, cell-based assays are necessary for high content analysis of proteolytic activities and for high content screening for novel protease modulators.
Fluofarma has developed novel, sensitive and cost-effective cellular probes for quantifying proteolytic activity in intact cells using membrane-bound fluorescent probes. These differential anchorage probes (DAP) combine a membrane anchorage domain, a protease cleavage peptide and a fluorescent protein such as GFP (Figure 1). Upon protease cleavage, the fluorescent tag is released from the anchor. Using this feature, proteolytic activity is deduced from the extent of the probe's retention observed after plasma membrane permeabilisation and washout of the cleaved fraction Fluofarma has developed several DAPs for the analysis of pivotal proteases such as caspase 3, caspase 7, calpain or gamma scretase, in several cell lines.
Thanks to the versatility of DAP technology, Fluofarma is further expanding its portfolio of cell-based assays with additional key proteases involved in tumour progression and degenerative diseases. Fluofarma's DAPs are suitable for various high content screening platforms including confocal microscopy, laser scanning microscopy, and flow cytometry.
Fluofarma is a privately held contract research organisation providing unique expertise services in cell-based assays development and high content analysis for the validation of therapeutic targets and for the characterisation of drug candidates.