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, 9 (9), 690-701

Emerging Principles in Protease-Based Drug Discovery


Emerging Principles in Protease-Based Drug Discovery

Marcin Drag et al. Nat Rev Drug Discov.


Proteases have an important role in many signalling pathways, and represent potential drug targets for diseases ranging from cardiovascular disorders to cancer, as well as for combating many parasites and viruses. Although inhibitors of well-established protease targets such as angiotensin-converting enzyme and HIV protease have shown substantial therapeutic success, developing drugs for new protease targets has proved challenging in recent years. This in part could be due to issues such as the difficulty of achieving selectivity when targeting protease active sites. This Perspective discusses the general principles in protease-based drug discovery, highlighting the lessons learned and the emerging strategies, such as targeting allosteric sites, which could help harness the therapeutic potential of new protease targets.


Figure 1
Figure 1. Tetrahedral intermediates formed during peptide cleavage by proteases
All proteases accelerate the formation of a transition state in peptide-bond hydrolysis. This state, termed the tetrahedral intermediate, is a prerequisite for peptide-bond scission in all protease mechanistic classes. In the case of serine (Ser), cysteine (Cys) and threonine (Thr) proteases, the tetrahedral intermediate involves a stable covalent bond to the enzyme’s catalytic nucleophile (Nuc), whereas metalloproteinases and aspartic acid (Asp) proteases use a non-covalent acid–base mechanism. The tetrahedral intermediate is difficult to simulate; protein engineers and chemists have yet to invent or evolve an independent chemical structure or fold that can efficiently catalyse peptide-bond cleavage. There are examples in which catalytic antibodies, for example, have been evolved to efficiently hydrolyse activated esters, but these ester bonds are extremely different from the resonance-stabilized peptide bond. This is a surprising gap in our knowledge and remains fertile ground for future work.
Figure 2
Figure 2. Active site, exosite and allosteric site
Three proteases of known structure have been chosen to illustrate the components identified in many (but not all) proteases that define their specificity and their control of activity. Surfaces of sentrin-specific protease 2 (SENP2) (Protein Data Bank code 1TGZ125) (a) and thrombin (Protein Data Bank code 1EB1 (REF. 126)) (b) with substrate or inhibitor residues contacting the active site cleft (shown in red sticks) and with substrate or inhibitor residues contacting the exosite (shown in green sticks) are shown. In SENP2, the exosite is composed of a large disperse region that occupies the bulk of the small ubiquitin-like modifier (SUMO) substrate, which is required for directing specificity and enhancing activity. By contrast, the thrombin exosite consists of a relatively small cationic patch that is required for efficient catalysis of fibrinogen and for binding to the inhibitor hirudin. The surface of Vibrio cholerae RtxA toxin (Protein Data Bank code 3GCD127) (c) centres on the allosteric natural small-molecule activator inositol hexakisphosphate (shown in white), with the active site bound peptide (shown in red) underlying a loop in the translucent surface. In the case of SENP2, the exosite is potentially quite large, complicating small-molecule control. However, in thrombin and RtxA the exosite or the allosteric site is composed of surfaces compatible with small-molecule targeting.
Figure 3
Figure 3. Probing protease active sites
In standard protease notation, substrates are cleaved between the P1 (amino-terminal) and P1′ (carboxy-terminal) residues, with P1,2,3 … n and P’1,2,3 … n residues increasing towards the N terminus of the protein and towards the C terminus respectively. The corresponding pockets on the protease that accept the substrate side chain are designated S or S’ accordingly. The schematic representation of the active site cleft of proteases shows the prime S1′-S3′ pockets accommodating specific amino acid side chains identified on the C-terminal side of the scissile bond and unprimed sites S1–S4 identified on the N-terminal side of the scissile bond (indicated by scissors). Shown also are the types of chemical moiety that can be used to investigate protease specificity either as individual substrates or as combinatorial libraries. The application of positional scanning substrate libraries allows exploration of the individual pockets of the protease by synthesis of a set of sublibraries. These generally consist of natural amino acids linked to a fluorogenic reporter, or, for a wider exploration of the catalytic cleft, of peptides that contain fluorophores and a quencher. The cleavage preference for each sublibrary is determined from fluorescence yields generated when the fluorophore is separated from the quencher by cleavage of the peptide bond.

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