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The Fluofast® Protease Assay
The Fluofast® protease assay designed and patented by Pharmaleads is a unique and non radioactive method to discover an optimal substrate for all types of proteases. The Fluofast® protease assay combines very high sensitivity and speed required for protease detection and high-throughput screening for inhibitors or activators. Pharmaleads Fluofast® technology applies for identification of substrates for any given protease.
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Main features of the Fluofast® Protease assay :
· Direct fluorescence measurement using purified enzymes or tissues or cellular homogenates.
· Homogeneous one step assay without washings.
· Detection of both activators and inhibitors, with no screening effect.
· Intense fluorescence increase (from 50 to 550 fold) following protease-induced hydrolysis (see graph besides)
· Rapid characterisation of the best-suited substrate within a library of 5000 molecules.
· To be used with all commercially available spectrofluorimeters and multi-well plate readers.
· Early indication on enzyme sub-sites preference for optimal inhibitor design.
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..N. Luciani et al., Biochem. J.,356, 2001.
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Principle of the assay :
This original concept is based on the ICIFQ (Internal Collision Induced Fluorescence Quenching) technology.
The Pyrenylalanine (Pya), one of the most powerful fluorogenic compound available, is introduced within the sequences of a library of peptide substrates and its fluorescence is strongly quenched by the proximity of the polar amino-acid, para-nitrophenylalanine (Nop).
The activity of a protease is revealed by the cleavage of the specific sequence located between the Pya and Nop groups, liberating the extremely intense fluorescence of the Pya compound.
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The library of 5000 substrates was obtained by introducing n amino acids, n ranging from 0 to 4, between the Pya and Nop compounds. The amino acids (X) are selected among a series of ten natural amino-acids.
The PHARMALEADS technology has been applied to rapidly detect substrates for all kinds of proteases belonging to metallo-, serine-, cystein- and aspartyl- protein classes. As an example in the figure besides, a restricted library (n=2) has been used to detect potential substrates for all classes of proteases. The intensity of the spots reflects the presence of cleavage(s) in each group of ten potential substrates. The identification of the metabolites by mass spectrometry gives the structure of the substrate hydrolysed. In case several substrates are hydrolysed, the choice of the best candidate will be made by comparing kinetic constants values and specificity.
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