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Targeting proteases: successes, failures and future prospects

Key Points

  • Proteases catalyse the hydrolyis of peptide bonds in proteins, often in a very precise way, and are thereby involved in the control of a number of important physiological processes including cell-cycle progression, DNA replication, cell proliferation and cell death, as well as the immune response

  • Protease signalling varies from a simple direct cleavage of a substrate to a complex cascade organization or a protease network, and requires tight regulation

  • Excessive proteolytic activity often leads to disease but can be prevented by blocking the appropriate proteases, which has been explored therapeutically since the 1950s.

  • Angiotensin-converting enzyme (ACE) inhibitors, which were introduced in 1981 for the treatment of various cardiovascular diseases (hypertension, heart failure, heart attack and so on), are still the major blockbusters among protease inhibitors on the market. On the other hand, various broad-spectrum matrix metalloprotease (MMP) inhibitors failed in advanced clinical trials for cancer and rheumatoid arthritis treatment in the 1990s because of various drawbacks.

  • Identification of endogenous protease substrates and other physiological protease ligands is a key issue in understanding protease signalling pathways and an essential part of the identification and validation of protease targets

  • Therapeutic inhibition of validated protease targets can be achieved either by large or small molecules. Large-molecule approaches include protein-type inhibitors that mimick physiological inhibitors and neutralizing antibodies. The development of small-molecule inhibitors, however, is by far the most popular approach. An ideal inhibitor would be a non-covalent, reversible inhibitor with excellent selectivity, good bioavailability and no side effects. The major issues in inhibitor design are still bioavailability and toxicity.

  • The most advanced inhibitors in clinical trials are the renin inhibitors aliskiren (SPP100) for the treatment of hypertension and end-organ damage for which an NDA was filed in 2006, and the dipeptidyl peptidase IV (DPP IV) inhibitors sitagliptin (MK-0431) and vildagliptin (LAF 327) for the treatment of type 2 diabetes, for which NDAs were also filed in 2006. Balicatib (AAE581), the most advanced among the cathepsin K inhibitors for osteoporosis treatment, successfully passed Phase II trials in 2005. Diabetes type 2 and osteoporosis are completely new therapeutic areas, which is encouraging for the future.

  • Proteases, such as kallikrein 3 (prostate-specific antigen) and plasminogen activator, are important diagnostic and prognostic disease markers.

  • The future of protease-based drug discovery efforts probably lies in the cardiovascular, inflammatory, infectious disease, cancer and neurodegeneration areas.

Abstract

Until fairly recently, proteases were considered primarily to be protein-degrading enzymes. However, this view has dramatically changed and proteases are now seen as extremely important signalling molecules that are involved in numerous vital processes. Protease signalling pathways are strictly regulated, and the dysregulation of protease activity can lead to pathologies such as cardiovascular and inflammatory diseases, cancer, osteoporosis and neurological disorders. Several small-molecule drugs targeting proteases are already on the market and many more are in development. The status of human protease research and prospects for future protease-targeted drugs are reviewed here, with reference to some key examples where protease drugs have succeeded or failed.

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Figure 1: Protease basics.
Figure 2: The renin–angiotensin system and drug discovery.
Figure 3: Regulation of protease activity.
Figure 4: New protease inhibitors in development.

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Acknowledgements

Research in the Turk laboratory is supported by grants from Slovene Research Agency. I wish to thank primarily G. Salvesen for numerous discussions, similar way of thinking and permission to use information and figures from his summary of the Horizon Symposium 'Signalling scissors: new perspectives on proteases' (see Further information). Also thanks to H. P. Nestler for providing most of the data for Table 1, G. Guncˇar for help with figure preparation, D. Turk and V. Turk for valuable discussions and critical reading of the manuscript, and D. Deaton, D. Percival, E. Altmann, R. Thurmond, N. Borkakoti, U. Grabowska, B. Gerhartz, V. Dive, and many others for sharing their data with me. I would also like to acknowledge the authors of the other 300,000 papers found in Medline using protease/proteolysis/proteinase as keywords, whose work contributed to our current understanding of proteolysis but could not have been cited here because of space limitations.

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DATABASES

OMIM

Alzheimer's disease

Paget's disease

Pycnodysostosis

Rheumatoid arthritis

Type 2 diabetes

FURTHER INFORMATION

MEROPS database

Signalling scissors: new perspectives on proteases

Glossary

Zymogen

A zymogen or pro-enzyme is an inactive enzyme precursor. A zymogen requires a biochemical change (such as a hydrolysis reaction revealing the active site, or changing the conformation to reveal the active site) for it to become an active enzyme.

Autocatalytic

The proteolytic conversion of a zymogen protease molecule into its mature form by molecules of the same kind.

Extracellular matrix degradation

The extracellular matrix are non-celullar components of tissues, and are primarily composed of various glycoproteins, proteoglycans and hyaluronic acid. In various disease states such as cancer and osteoarthritis, ECM is degraded by various proteases, including matrix metalloproteases and the cathepsins.

Positional scanning library

A combinatorial chemistry approach in which individual positions in the molecule are kept defined with the other positions being degenerate to yield 'positional libraries'. These compound mixtures are then tested. The most active moieties of the defined (and therefore known) positions are subsequently combined to yield the screening result, the active molecule.

Mature protease

The catalytically active form of a protease that results from proteolytic processing of its zymogen (inactive pro-enzyme).

Warhead

A reactive functional group, which covalently binds to amino-acid residues of the target enzyme.

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Turk, B. Targeting proteases: successes, failures and future prospects. Nat Rev Drug Discov 5, 785–799 (2006). https://doi.org/10.1038/nrd2092

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