Fluorescence Intensity

Most biochemical assays used to determine the potency of inhibitors against (1) aminopeptidases and (2) endopeptidases with minor contributions to the substrate binding efficiency by the S' site are based on the measurement of fluorescence intensity (FI). The FI readout principle is shown in Figure 2.2. The dynamic range of the FI readout basically scales with the fluorescence quantum yield, that is, the efficiency of fluorescence emission of the dye label. For the FI readout, peptide substrates with fluorogenic groups such as acetylmethoxycoumarin (AMC), 7-amino-4-trifluoromethyl

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.

coumarin (AFC), and rhodamine 110 (Rh110) attached to the C terminus are frequently used. Table 2.1 summarizes the key characteristics of this selection of fluorophores.

Dipeptidylpeptidases like DPPIV cleave the peptide bond between the amino acids in P1 and P1' positions with the label attached to the P1' position. Aminopeptidases cleave the pseudo peptide bond between the amino acid in P1 position (= C terminus) and the fluorophore (Figure 2.2). This is possible with high catalytic efficiency (kcJKM), because the aminopeptidases primarily recognize and bind to the N termini of their substrates. The dye attached to the C terminus does not alter the protease-substrate interaction. The cleavage of the labeled peptide results in an increase in total fluorescence due to the reconstitution of the fluorophore system. Historically, AMC was most frequently used as a fluorogenic group in biochemical protease assays with mono-labeling of the substrate. Three publications on cathepsin G assays serve as examples of 20 years of progress in assay design based on FI as the readout vehicle (Tanaka et al., 1985; Rehault et al., 1999; Attucci et al., 2002).

AMC is still a popular dye today. According to our experience, the IC50 values determined for protease inhibitors in AMC-based protease assays may be biased and misinterpreted due to the interference of label and compound autofluorescence characteristics (Figure 2.3). In the worst case, this can lead a drug discovery program in a wrong direction with respect to prioritization of compound classes. At the excitation and emission wavelengths of 350 and 500 nm, respectively, used for AMC, many compounds also display fluorescence characteristics.

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