What is Proteases?

Proteases are enzymes that are able to cleave or degrade other proteins. The functional and regulatory capacities of proteases vary greatly and include, but are not limited to, blood clotting, extracellular matrix degradation, activation of apoptosis, protein degradation, and viral protein processing.

Overview

Proteases (also known as proteinases) cleave peptide bonds in proteins by nucleophilic attack of the peptide carboxyl group. The general term “protease” also encompasses “peptidases” which are active only against small peptide substrates. For example a “carboxypeptidase” will catalyze cleavage of one amino acid from the carboxyl terminus of a peptide. Generation of the nucleophile varies across proteases, therefore proteases are generally categorized by their catalytic mechanism of action:

  • Asparagine
  • Aspartyl
  • Cysteine
  • Glutamic
  • Metallo
  • Serine
  • Threonine

Since the mechanism of action of a protease does not dictate its biological function, and proteases with similar catalytic mechanisms of actions can be targeted by related inhibitors, structurally similar drugs can be used to treat a variety of diseases. Therefore this categorization is helpful for thinking about proteases as drug targets.

Existing Products

There are numerous protease inhibitors that are approved or in late stage clinical development for a variety of diseases. These products include inhibitors for five of the seven classes of proteases.

Protease Class Target Name Development Status
Metalloprotease Angiontensin converting enzyme (ACE) FDA approved for hypertension
Aspartyl protease Renin FDA approved for hypertension
HIV-1 protease FDA approved for HIV infection
Threonine protease Proteasome FDA approved for multiple myeloma
Serine protease Dipeptidyl aminopeptidase 4 (DPP4) FDA approved for diabetes
HCV NS3/4A protease Phase III for HCV infection
Cysteine protease Cathepsin K Phase III for osteoporosis

Protease Inhibitors as Non-Neglected Tropical Disease Therapeutics

There are several diseases that are not related to neglected tropical diseases for which proteases have been targeted, including:

  • Cancer
  • Cardiovascular disease
  • Diabetes

Proteases play numerous roles in cancer pathogenesis. They promote cancer by degrading extracellular matrix surrounding tumors, thus promoting metastasis of cancer to other organs (primarily via cysteine proteases and metalloproteases). Proteases also play an essential role in cancer treatment as a key step in the activation of apoptosis. Certain cancer cells also appear to be more sensitive to the inhibition of essential proteases than healthy cells.  This was demonstrated with the proteasome inhibitor Velcade (bortezomib) (Millennium/Takeda), which was first approved for the treatment of multiple myeloma in 2003.

In cardiovascular disease, proteases are best known for their role in hypertension. Angiotensin is a peptide that has been shown to cause blood vessel constriction, thus increasing blood pressure and causing hypertension, a known risk factor for heart attacks and other cardiovascular disease. Angiotensin levels are regulated by the renin-angiotensin system which includes two key proteases: renin (aspartyl protease) and angiotensin converting enzyme (ACE; metalloprotease). A core component of hypertension treatment is ACE inhibition which blocks conversion of angiotensin I to angiotensin II, the form of angiotensin responsible for vasoconstriction.

The serine protease dipeptidyl amino peptidase 4 (DPP4) is a protease involved in glucose regulation and is a therapeutic target for the treatment of diabetes. The gut hormone GLP-1 is an important signal for insulin release. The protease DPP4 inactivates GLP-1 thus lowering insulin release and increases blood glucose levels. Therefore, inhibition of DPP4 can promote insulin release and lower blood glucose in patients with diabetes. The first DPP4 inhibitor for the treatment of diabetes, sitagliptin (Merck), was approved by the U.S. Food & Drug Administration (FDA) in 2006.

Proteases play important roles in the pathogenesis of other non-neglected tropical diseases. Protease inhibitors are in development for the treatment of many of these other disorders.

Protease Inhibitors as Neglected Tropical Disease Therapeutics

HIV infection (AIDS) was the first infectious disease affecting both resource rich and poor regions of the world for which a protease inhibitor was successfully developed as a therapeutic. The HIV-1 protease is an aspartyl protease that cleaves the polyprotein produced by the replicating virus into various component proteins of the HIV virion. Cleavage of the polyprotein is essential for viral replication and assembly. There are currently 11 different protease inhibitor products that are FDA approved for the treatment of HIV.1

Protease drug development for HIV has benefited from the recycling of chemical compound libraries originally developed for non-infectious disease proteases with related mechanisms of action. For example, early lead compounds for the HIV-1 protease were identified by screening aspartyl protease inhibitors originally developed for human renin for the treatment of hypertension.

References

  1.  FDA, Antiretroviral drugs used in the treatment of HIV infection, available here.

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Pipline

Analysis

Proteases across multiple classes have been validated as therapeutic targets for a variety of diseases. As a target class, proteases benefit from an extensive body of existing research into the understanding of their catalytic mechanisms, substrate specificity, and structures. Additionally, protease drug development benefits from the recycling of chemical compound libraries between protease targets with similar mechanisms of action.

The relative strengths, weaknesses, opportunities, and risks for protease inhibitors that are currently in development for neglected tropical diseases are summarized here.

  Strengths Weaknesses Opportunities Risks
Protease inhibitors
Relevant neglected tropical diseases: 

HIV (multiple products, on market) 

Chagas (K777, pre-clinical)

Malaria (Falicpain 2/3 inhibitors, discovery; DPAP1 inhibitors, discovery)

Tuberculosis (multiple proteases, discovery)
Proteases validated as targets for multiple infectious and non-infectious diseases

Target-associated drug development tools (i.e., compound libraries, high-throughput screening assays, and crystal structures) are widely available
 
Many proteases have orthologs in the host, so to avoid toxicity inhibitors may be required to have selectivity over host proteases. However there are also several infections in which the target protease is more vulnerable in location to the protease inhibitor so even less selective drugs can be developed. Likely that many relevant compound libraries exist in industry/biotech

Numerous neglected tropical diseases have protease orthologs; however, validation of more of these orthologs as therapeutic targets is needed
 
All current projects are in early stages, so it may take a long time to validate proteases as drug targets for additional neglected tropical diseases in the clinic.

Beyond the ongoing protease inhibitor drug development programs for neglected tropical diseases, additional proteases have been genetically or chemically validated as potential therapeutic targets for these diseases. These validated targets represent the best opportunity for immediate application of existing small molecule libraries from non-neglected tropical disease protease inhibitor drug discovery programs.

Neglected Tropical Disease NTD Drug Targets by Protease Class
  Aspartyl Cysteine Metallo Serine Threonine
Chagas   Cruzain      
Dengue NS3 protease        
HAT   TbCatB, Brucipain, Rhodesian     TbProteasome
HIV       HIV-1 protease  
Malaria   Falcipain 3, DPAP1 & 3, Vivipain-4 (P. vivax) Falcilycin, PfA-M1, PfLAP, PfAPP PfSUB1, PfSUB2 PfProteasome
Tuberculosis     MtMetAP1c, PDF   MtProteasome

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Database Resources

Known proteases, protease inhibitors, and protease substrates are extensively cataloged in the MEROPS database1,2 and the Proteolysis Map (PMAP)3 database. Updates on advances in protease research are available through the International Proteolysis Society, a professional organization focused on understanding all aspects of protease research.4

Assays

Numerous assays are available to study protease activity and function. The most common are fluorogenic substrate assays that are amenable to high throughput screening. These assays can be performed on recombinant proteases or, in some cases, in complex proteomes.

Additional assays used to study protease function and activity include:5,6,7

  • Positional scanning substrate libraries: For proteases with unknown substrate specificity, diverse libraries of substrates that use fluorescent readout to profile substrate specificity of recombinantly expressed proteases are available
  • Activity-based probes: Irreversible protease inhibitors with tags that can be used for visualization or monitoring of protease activity in gel-based, live cell, or high throughput screening assays
  • Mass spectrometry identification and profiling of protease substrates: Numerous methods to identify protease substrates in complex proteomes, primarily through comparison of proteomes with and without active protease, are available.

References

  1. Wellcome Trust Sanger Institute, MEROPS: the Peptidase Database, available here
  2. Rawlings ND et al. (2010) “MEROPS: the peptidase database.” Nucleic Acids Res 38: D227-D233.
  3. The Proteolysis Map (PMAP), available here.
  4. International Proteolysis Society, available here
  5. Drag M and Salvesen GS (2009) "Emerging principles in protease-based drug discovery." Nature Reviews Drug Discovery 9: 690-701.
  6. Diamond SL (2006) "Methods for mapping protease specificity." Current Opinion in Chemical Biology 11: 46-51.
  7. Agard NJ and Wells JA (2009) "Methods for the proteomic identification of protease substrates." Current Opinion in Chemical Biology 13: 503-509.

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