Protease assays

Kohl, T., Heinze, K. G., Kuhlemann, R., Koltermann, A. and Schwille, P. (2002). A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc. Natl Acad. Sci. USA 99, 12161-6. 

A succinylated casein derivative that has nearly all its amines blocked can be used as a substrate in protease assays (Hatakeyama et al., 1992). As the casein is degraded by a protease, free amines are created from a-chain cleavage and release of a-amino groups. The creation of... 

Fig. 10 Protease assay using beads with emissive polymer and avidin [87] cartoon. Emissive polymer is represented by green lines, quenched polymer by grey...

Meldal et al. developed a novel protease assay based on the long-range resonance energy transfer (FRET) [25] fluorescence quenched (EQ) pair 3-nitrotyro-sine/2-aminobenzoic acid. This served to characterize enzyme specificity by direct visual inspection of the resin beads (33). 

Sarath, G., Zeece, M.G., and Penheiter, A.R. 2001. Protease assay methods. In Proteolytic Enzymes A Practical Approach (R. Benyon and J.S. Bond, eds.) pp. 45-76. Oxford University Press, Oxford. 

In a general serum stability assay, the peptide is subjected to human serum (see Note 3) at realistic temperature conditions and incubated for various time intervals, comparable to traditional protease assays. The reactions are stopped by TCA or ethanol, precipitating larger serum proteins while leaving peptides... 

Kinase substrates can become resistant to the actions of proteases due to their phosphorylations. Thus, the fluorescence quench assays (described in Chapter 2 covering protease assays) can be used to measure kinase activity. The assays can be viewed as coupled because they require a second enzyme to convert a product or substrate into a detectable signal. With kinase assays, the formation of phosphopeptide inhibits the protease action on the peptide and the signal remains quenched and therefore decreased (Rodems et al., 2002). Inhibiting the kinase results in increases in protease sensitivity and in signal. 

Fluorescence-Based Biochemical Protease Assay Formats... 

In the second section of this chapter, strategies to identify substrates for biochemical protease assays are discussed. Section 2.3 focuses on theoretical and practical aspects of various fluorescence-based readouts for biochemical protease assays. Finally concrete experiments for the determination of enzyme kinetics relevant for the development of robust and sensitive biochemical protease assays are summarized in Section 2.4. Altogether this chapter offers guidelines for the development of biochemical protease assays for the purpose of protease inhibitor-directed drug discovery. 

Fluorescence-based readouts build by far the most important basis of protease assays for HTS and compound profiling in the drug discovery industry today. These assay formats became increasingly popular in the 1990s (Burbaum and Sigal, 1997 Silverman et al., 1998). The basics of fluorescence are very well described in detail by Lakowicz (2006). This discussion will be restricted to biochemical protease assays in homogeneous formats based on fluorescence readouts that are suitable for HTS and automated compound profiling. 

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. 

Fluorophores Frequently Used for Protease Assays Based on FI Readout... 

Abz was combined with a broad variety of non-fluorescent acceptors such as p-nitrobenzyl for leucine aminopeptidase (Carmel et al., 1977), pNA for trypsin (Bratanova and Petkov, 1987), 4-ni-trophenylalanine [Phe(NC>2)] for HIV protease (Toth and Marshall, 1990), and V-(2,4-di n itrophenyl) ethylenediamine (EDDnp) for thermolysin and trypsin (Nishino et al., 1992). Lecaille et al. (2003) described a FRET quench assay based on a specific substrate for cathepsin K labeled with Abz and EDDnp. This substrate is not cleaved by the other Cl cysteine cathepsins and serine proteases in contrast to methoxycoumarin (Mca)-based substrates described earlier (Aibe et al., 1996 Xia et al., 1999) and merely covered the non-primed site of the scissile bond. The 5-[(2-aminoethyl)amino] naphthalene-l-sulfonic acid (EDANS) compound is a second example of a fluorescence donor historically used for many FRET quench-based protease assays, e.g., in combination with tryptophan as a quencher in an ECE activity assay (Von Geldren et al., 1991). The FRET-1 example in Table 2.2 shows the typical dynamic range that can be achieved with an EDANS/DABCYL-based assay. 

The same publication indicated that fluorescence lifetimes of compounds from the compound collection of Pfizer classified as problematic due to their autofluorescence characteristics resulted in false positive results in many FI-based assays. For most compounds, the fluorescence lifetimes were below 1 ns. Thus, FLT measurements with a reporter fluorophore displaying a lifetime significantly longer than 1 ns are suitable for application in protease assays for compound testing. However, for applications under initial velocity conditions with a substrate turnover below 20%, fluorophores with lifetimes of a few nanoseconds are still critical because the dynamic range of the assay is then too low with lifetimes below 1 ns. 

Protease assays based on the FLT of PT14 are especially attractive due to the long lifetime of the dye of 14 ns that allows the discrimination of the lifetimes of short-lived fluorescent compounds from that of PT14. Thus, the rate of false-positive and -negative results can be reduced. The major... 

The Km value also affects the substrate concentration employed in the assay. The assay is more sensitive to weak competitive inhibitors when working at low substrate concentrations. In drug discovery, protease assays are usually run with substrate concentrations at or below the KM value, because the focus is mainly on the identification and characterization of competitive inhibitors (Cheng and Prusoff, 1973 Copeland, 2005). The turnover number kcat is a first-order rate constant that defines the maximum number of substrate molecules converted to product per time unit under saturating conditions (i.e., at high excess of substrate). 

The development of protease assays is very straightforward if (1) the substrate recognition sequence for the protease of interest is known and (2) short peptides can be used as substrates. Extensive amounts of information about substrates are available from electronically searchable databases in the public domain. In addition, several strategies to experimentally identify substrates for proteases of unknown specificity have been described. The most recent method is the so-called PICS technology that covers all the sequences relevant in a human proteome. The MS/MS-based identification of the cleavage products and the subsequent identification of single substrate peptides... 

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