Protease fluorescence intensity

In a different approach, fluorescence-based DNA microarrays are utilized (88). In a model study, chiral amino acids were used. Mixtures of a racemic amino acid are first subjected to acylation at the amino function with formation of A-Boc protected derivatives. The samples are then covalently attached to amine-functionalized glass slides in a spatially arrayed manner (Fig. 10). In a second step, the uncoupled surface amino functions are acylated exhaustively. The third step involves complete deprotection to afford the free amino function of the amino acid. Finally, in a fourth step, two pseudo-Qn nX. om.Qx c fluorescent probes are attached to the free amino groups on the surface of the array. An appreciable degree of kinetic resolution in the process of amide coupling is a requirement for the success of the ee assay (Horeau s principle). In the present case, the ee values are accessible by measuring the ratio of the relevant fluorescent intensities. About 8000 ee determinations are possible per day, precision amounting to +10% of the actual value ((S(S). Although it was not explicitly demonstrated that this ee assay can be used to evaluate enzymes (e.g., proteases), this should in fact be possible. So far this approach has not been extended to other types of substrates. 

FIGURE 2.2 Fluorescence intensity readout principle. In the intact peptidic substrate (amino acids symbolized by X, Y, and Z) labeled with a fluorophore at the C terminus, the intensity of fluorescence emission (light gray arrow) after excitation (dark gray arrow) is low. Through the cleavage of the substrate between the C terminal amino acid (Z) and the fluorophore by a protease, the intensity of fluorescence emission is strongly enhanced. An increase of fluorescence intensity over time dependent on the enzymatic velocity is observed. 

FIGURE 2.3 Dose-response curves for protease inhibitor with autofluorescence characteristics, (a) Result of standard fluorescence intensity-based assay employing an AMC-labeled substrate at a concentration of 2 pM. Profiling data could not be obtained due to the interference of the compound s fluorescence with the readout, (b) Result obtained from assay employing a RhllO-based substrate at a concentration of 0.5 pM. The profiling data (IC50 value of 335 nM and Hill coefficient of 1.0) were obtained for the depicted data set. 

Hassiepen, U. et al. 2007. A sensitive fluorescence intensity assay for deubiqitinating proteases using ubiquitin-rhodamine 110-glycine as substrate. Anal. Biochem. 371, 201-207. 

As mentioned above, assay readouts based on fluorescence are probably the most commonly used of all HTS detection techniques. A number of different flavors of fluorescence-based assays exist. The simplest one, called fluorescence intensity (FLINT) was used, for example, to assay for inhibitors of a proteolytic enzyme that releases a fluorescent component upon cleavage of a specially designed substrate, as shown in Figure 6.3. In this case the enzyme of interest is Cathepsin K, a cysteine protease critical to bone resorption, inhibitors of which are of interest to fight osteoporosis. ... 

Another example of the incorporation of two unnatural photonic groups into a single protein was shown by Hecht and coworkers. Dfliydrofolate reductase (DHFR) was engineered with a fusion peptide at its N-terminus that contains the quencher (7VP-dabcyl-l,2-diaminopropionic acid) and fluorophore (7-azatryptophan) flanking an HIV-1 protease cleavage site. In vitro expression involved use of an amber suppressor tRNA and a four-base anticodon tRNA. The resulting DHFR fusion complex exhibited an increase in fluorescence intensity upon treatment with HIV-protease. 

Proteases are one of the largest families of enzymes and are involved in a multitude of vital processes. Due to their biological relevance and diversity, multiple fluorescent reporters monitoring their activity have been designed and successfully applied in vitro and in vivo [112-114]. Standard small molecule FRET probes for proteases consist of an amino acid sequence flanked by a FRET pair, consisting of two fluorophores or one fluorophore and a quencher molecule. Upon cleavage of the peptide sequence, the emission of the donor fluorophore is dequenched and the intensity increases whereas the emission of the acceptor decreases and vanishes more or less completely in those cases where the acceptor is fluorescent (see Fig. 6.11). 

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