Fluorophores Frequently Used for Protease Assays Based on FI Readout

Fluorophore Excitation Wavelength (nm) Emission Wavelength (nm) Reference

AMC ACC AFC Rh110

350 350 400 485

500 450 505 535

Okun et al., 2006 Harris et al., 2000 Gurtu et al., 1997 Grant et al., 2002

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 |M. 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 Rh110-based substrate at a concentration of 0.5 |M. The profiling data (IC50 value of 335 nM and Hill coefficient of 1.0) were obtained for the depicted data set.

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 |M. 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 Rh110-based substrate at a concentration of 0.5 |M. The profiling data (IC50 value of 335 nM and Hill coefficient of 1.0) were obtained for the depicted data set.

Red-shifted dyes are much less prone to interfering artifacts and thus result in significantly fewer false-positive and false-negative hits in HTS or compound profiling (Turconi et al., 2001a; Banks et al., 2000). Therefore, fluorophores such as Rh110 are applied more frequently today (Grant et al., 2002; Graziano et al., 2006). In our experience, Rh110 is the most suitable dye for protease activity assays to determine inhibitor potency based on the FI readout. The fluorescence of Rh110 is usually excited at 488 nm and the emission is detected at 535 nm. Commercially available peptides carrying Rh110 labels are symmetrically to-substituted in most cases, e.g., XYZ-Rh110-ZYX with the amino acid sequence XYZ at positions P3 to P1, because the synthesis of these substrates is easier than for asymmetric peptides. Therefore most Rh110-based protease assays are developed using symmetric peptidic substrates.

The application of asymmetric peptides is much less frequently described (Cai et al., 2001). The production of asymmetric Rh110-labeled to-substituted peptides is much more difficult because the synthesis requires significantly more steps than required for symmetrically to-substituted peptides. The critical step is the synthesis of the mono-substituted intermediate product Rh110-ZYX with low yield in most cases (personal communication). However, due to the fact that a strong fluorescence increase is only observed after the fluorophore has been cleaved off from both peptide chains of the symmetrically bis-substituted substrate (two-step proteolysis), the kinetics of the enzymatic reaction can be followed more accurately and easily with peptides comprising only one copy of the scissile bond.

To improve the solubility of asymmetric peptides, it is advisable to use the non-primed site recognition sequence of the protease combined with a Rh110 molecule linked to an acidic amino acid (e.g., glutamate) via the 7-carbonyl function, i.e., XYZ-Rh110-yGlu. In some cases, this may lead to a slight increase of the KM value or a slight reduction of the catalytic efficiency (kcJKu) in comparison to the analogous peptidic substrate mono-labeled with AMC and without yGlu. In general, the catalytic efficiency determined with C terminally labeled peptides tends to be lower in comparison to unlabeled substrates because of the location of the fluorophore at the scissile bond, substituting the natural amino acid in position P1'. The fluorophores are bulky aromatic groups differing from the natural amino acids. Moreover, the amide bond between the C terminal amino acid and the fluorophore differs from the usual peptide one.

As a rule, substrate concentrations of 100 to 500 nM for Rh110 and concentrations of 1 to 5 ^M for AMC are sufficient for the development of robust assays suitable for HTS and high-throughput

TABLE 2.2

Dynamic Ranges for Various Fluorescence-Based Biochemical Protease Assay Formats

TABLE 2.2

Dynamic Ranges for Various Fluorescence-Based Biochemical Protease Assay Formats

Reference

Substrate Sequence

Protease

A Signal

FI-1

Suc-L-L-V-Y i AMC

Chymase

fdyn - 250

FI-2

Suc-L-L-V-Y i Rh110-^E

Chymase

fdyn - 250

FRET-1

Ac-R-E(EDANS)-E-V-L-F-Q i G-P-K(DABCYL)-R-NH2

HRV 3C protease

fdyn - 40

FP-1

BTN-T-T-R-P-G-S-G-L-T-N-I-K-T-E-E-I-S-E-V-N-L i D-A-

BACE-1

160 mPU

E-F-R-H-D-K-TAMRA

FP-2

Ac-R-R-K(TAMRA)-L-L-V-Y i H-K(BTN)-OH

Chymase

330 mPU

FLT-1

Ac-E-F-K-P-I-L-W i R-L-G-C(PT14)-E-NH2

Kallikrein 7

5.5 ns

FLT-2

W-P i S-G-T-F-T-K-C(PT14)-NH2

DPP IV

5.8 ns

FLT-3

C(PT14-ME)-G-G i W-OH

Carboxypeptidase A

9 ns

FLT-4

Ac-C(PT14)-V-P-R i A-W-E-NH2

Thrombin

3.7 ns

FLT-5

PT14-D-E-V-D i W-E-NH2

Caspase-3

4.8 ns

All data obtained with Tecan Ultra Evolution MTP reader. The following excitation and emission wavelengths were used: EDANS and AMC: 350 and 500 nm; Rh110: 485 and 535 nm; TAMRA: 535 and 595 nm; PT14: 405 and 450 nm. I = primary cleavage site confirmed by MS. AMC = aminomethylcoumarin. Rh110 = rhodamine 110. gE = glutamic acid attached to Rh110 via its carbonic acid in side chain. EDANS = fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid. DABCYL = 4-(4-dimethylaminophenylazo)benzoic acid quencher. BTN = biotin. PT14 = acridone-based fluorescence lifetime label.

All data obtained with Tecan Ultra Evolution MTP reader. The following excitation and emission wavelengths were used: EDANS and AMC: 350 and 500 nm; Rh110: 485 and 535 nm; TAMRA: 535 and 595 nm; PT14: 405 and 450 nm. I = primary cleavage site confirmed by MS. AMC = aminomethylcoumarin. Rh110 = rhodamine 110. gE = glutamic acid attached to Rh110 via its carbonic acid in side chain. EDANS = fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulphonic acid. DABCYL = 4-(4-dimethylaminophenylazo)benzoic acid quencher. BTN = biotin. PT14 = acridone-based fluorescence lifetime label.

compound profiling according to our experience. Signal-to-noise ratios are higher with Rh110 in comparison to AMC and AFC due to a much higher extinction coefficient, fluorescence quantum yield, and photostability (Liu et al., 1999). Thus assay systems with low KM values and low substrate concentrations will usually give best signal-to-noise ratios with Rh110 compared to AMC. The FI-1 and FI-2 examples in Table 2.2 give an impression of the high dynamic range that can be achieved with FI-based assays. Due to the high speed and ease of detection, the FI readout is ideally suited for high-throughput applications. The dyes and synthesis of labeled peptide substrates are affordable and thus allow cost-efficient operations. FI assays can easily be adapted to the 1536-well format. On the other hand FI assay results can suffer from fluorescence interference by the tested compounds, leading to false positive and false negative results.

FI is not frequently used as a readout for carboxypeptidases because the assay principle cannot be applied easily. In C terminally labeled peptide substrates, the primed site part of the car-boxypeptidase recognition sequence is missing, resulting in high KM values, incompatible with the development of robust protease activity assays. The same limitation of the FI assay principle is observed with endopeptidases in which amino acids on the primed site of the substrate (primarily P1') strongly contribute to the binding energy of the peptide.

Enzymatic assays for aminopeptidases can also be based on the readouts described below, which are applied primarily for endopeptidases and carboxypeptidases. These readouts use peptide substrates that span from the non-primed to the primed side.

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