Introduction

Proteases are enzymes catalyzing the hydrolysis of peptide bonds. They form one of the largest enzyme families encoded by the human genome, with more than 500 active members. Based on the different catalytic mechanisms of substrate hydrolysis, these enzymes are divided into four major classes: serine/threonine, cysteine, metallo, and aspartic proteases. In serine, cysteine, and threo-nine proteases, the nucleophile of the catalytic site is a side chain of an amino acid in the protease (covalent catalysis). In metallo and aspartic proteases, the nucleophile is a water molecule activated through the interaction with amino acid side chains in the catalytic site (non-covalent catalysis) (Gerhartz et al., 2002).

From the perspective of the substrate, the proteases are classified as endopeptidases and exo-peptidases according to the nomenclature by Barrett and MacDonald (1986). In endopeptidases, the substrate runs through the entire length of the active site and is cleaved at an internal peptide bond in the middle of its sequence (Barret, 2004). In exopeptidases, substrate binding is structurally constrained so that only one or two amino acid residues of the substrate can specifically bind to the protease. The exopeptidase reaction is defined as the cleavage near or at either end of the substrate molecule, commonly directed by the recognition of the free, charged amino (N) or carboxy (C) terminal groups of the substrate by the protease. The aminopeptidases bind and cleave their substrates from the N terminus. The carboxypeptidases bind and cleave their substrates from the C terminus.

Non-Primed side

Scissile bond

Non-Primed side

Primed side

FIGURE 2.1 Standard nomenclature for substrate residues and their corresponding binding sites on the protease. The subsites toward the N terminus of the substrate are called non-primed sites and are numbered S1 to Sn, beginning with S1 at the N terminal side of the scissile bond. The subsites toward the C terminus of the substrate are called primed sites and are numbered ST to Sn' beginning with ST at the C terminal side of the scissile bond. The substrate residues that the enzymatic subsites accommodate are numbered P1 to Pn and PT to Pn', respectively.

The surface of a protease that is able to accommodate one single amino acid side chain of the substrate sequence is called a subsite. The subsites toward the N terminus of the substrate are called non-primed sites and are numbered S1 to Sn beginning with S1 at the N terminal side of the scissile bond (Figure 2.1). The subsites toward the C terminus of the substrate are called primed sites and are numbered S1' to Sn' beginning with S1' at the C terminal side of the scissile bond. The substrate residues that the enzymatic subsites accommodate are numbered P1 to Pn and P1' to Pn', respectively (Berger and Schechter, 1976). The structures of the catalytic, non-primed, and primed sites play an important role for the specific recognition of a protein or peptide substrate by the protease.

Initially identified as participants in mammalian food digestion in the intestinal tract, proteases were later recognized to play a central role in many physiological and pathological processes such as cell proliferation, blood coagulation, blood pressure control, protein catabolism, neurodegeneration, bacterial and viral diseases, and inflammation and cancer (Turk, 2006). Therefore the strategy to achieve pharmacologically relevant inhibition of proteases has been an attractive drug discovery strategy since the 1950s and paid off with a broad number of protease-inhibiting drugs on the market today.

The most prominent drug target example from the carboxypeptidase group is the angiotensin-converting enzyme (ACE). It is a metalloprotease that acts as carboxy dipeptidase, cleaving two amino acids from the C terminus of angiotensin. ACE plays a central role in the treatment of hypertension with inhibitors on the market for more than 20 years. Prominent examples from the class of the endopeptidases playing an important role as drug targets are the aspartic renin and HIV proteases. Inhibitors of both proteases are on the market. Rasilez treats hypertension; ritonavir and saquinavir treat AIDS. Prominent examples of exopeptidases as drug targets are the dipeptidylpep-tidase IV (DPPIV) serine protease for the treatment of type 2 diabetes and the serine proteases of the blood coagulation cascade, especially thrombin and factor Xa for the treatment of thrombosis (Turk, 2006). Several drugs inhibiting these serine proteases are on the market already (DPPIV inhibitors Januvia and Galvus; fXa inhibitor Xarelto) or in clinical development. All three enzymes belong to the aminopeptidase group.

A major starting point for drug discovery in the pharmaceutical industry is the identification of low molecular weight inhibitors by high-throughput screening (HTS) campaigns. Large numbers of compounds, typically a million, are tested for their inhibitory potential on the drug target of interest. HTS is followed by compound validation and optimization activities during which inhibitors are modified to improve potency on the target and selectivity over other members of the same target class or family. Both HTS and follow-up activities require robust and sensitive assays.

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.

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