New High-Throughput Screening Protease Assay Based upon Supramolecular Self-assembly

We previously demonstrated that the supramolecular self-assembly of cyanines could be useful for developing fluorescent enzymatic assays. We took that concept a step further by synthesizing a covalent adduct of the tetrapeptide Asp-Glu-Val-Asp (DEVD) and a cyanine (DEVD-cyanine). The DEVD-cyanine due to its canonical sequence was recognized and hydrolyzed by the proteases, Caspase-3 and -7 in 96- or 384-microwell plate reactions. The catalytically liberated cyanine self-assembled upon scaffolds of carboxymethylamylose (CMA), carboxymethylcellulose (CMC), or a mixture of CMA and CMC resulting in a J aggregate exhibiting bright fluorescence at a 470 nm emission wavelength (optimum signal/background using excitation wavelengths of 415-440 nm). The fluorescence intensity increased with enzyme and substrate concentrations or reaction time and exhibited classical saturation profiles of a rectangular hyperbola. Saturation of the reaction was at 30 U/mL (1 microg/mL) Caspase-3 and 250 microM DEVD-cyanine. The reaction kinetics was linear between 1 and 20 min and saturated at 60 min. The affinity constant (Km) for DEVD-cyanine was approximately 23 microM, similar to those of previously reported values for other DEVD substrates of Caspase-3. Maximal fluorescence emission was observed by using a mixture of CMA and CMC scaffolds at 65 and 35 microM, respectively. The reaction kinetics of Caspase-7 executed in a 384-well plate was similar to the reaction kinetics of Caspase-3 conducted in a 96-well plate. We believe that this is the first demonstration of a cyanine liberated from a covalent adduct due to protease action, leading to supramolecular self-assembly and the detection of protease activity.