OUR UNDERSTANDING OF THE ROLE OF PROTEASES has evolved from protein-degrading enzymes purely responsible for food digestion and intracellular protein turnover to important signaling molecules involved in complex pathways.1,2
Proteases regulate a large number of biological processes, including cell cycle progression, proliferation, death, migration, and the immune system.1
Proteases account for approximately 2% of the human genome, with 500 to 600 proteases identified to date; it is estimated that proteases may represent 5% to 10% of the potential drug targets.1,2
Drugs targeting proteases encountered early successes, such as the widely used angiotensin-converting enzyme inhibitors1
or the proteasome inhibitor bortezomib.3
Matrix metalloproteinases (MMPs) were also considered as attractive cancer targets because of their involvement in angiogenesis and metastasis through degradation of the extracellular matrix (ECM). After encouraging preliminary results in cancer models, several matrix metalloproteinase inhibitors such as batimastat were progressed to clinical trials but eventually failed because of severe side effects and/or lack of efficacy.1,4-6
It was later discovered that MMPs promote tumor progression not only through ECM degradation but also through signaling functions.2
Although some MMP functions contribute to counter apoptosis, organize angiogenesis, and promote metastasis and tumor growth, thereby constituting targets for antitumor agents, some MMP functions also protect the host, for example, by organizing the innate immune response.2
This dual role of MMPs in cancers explains for a big part the failure of MMP inhibitors in clinical trials. It is therefore important for future drug development to distinguish MMPs that contribute to tumor progression (targets) from those necessary for host defense (antitargets).
Most published MMP inhibitors contain a strong chelating moiety such as a hydroxamic acid.7
This is because most of the binding energy to MMPs for these compounds is derived from the interaction between the chelating moiety and the ion in the active site.2,7
These agents therefore do not discriminate well between different MMPs2,7
or even between metalloproteinases (MPs).2,8
For example, an activity-based probe derived from the broad-spectrum MMP inhibitor ilomastat (GM6001; see ), which was in clinical development for patients with advanced cancer but later abandoned, was shown to target the nonrelated MPs neprilysin, leucine aminopeptidase, and dipeptidyl peptidase III.8
The observation that most existing MMP inhibitors are broad metalloprotease inhibitors (MPIs), when put in perspective with the absolute necessity to spare the antitarget MMPs, is key to explain their failure in clinical trials.
Panel of 8 Metalloprotease Inhibitors (MPIs)
Peptide deformylase (PDF) inhibitors have long been sought as antimicrobial agents. The discovery that actinonin inhibits PDF oriented research efforts toward peptidomimetic, hydroxamic acid-based inhibitors.9
Hence, the general structure of most current PDF inhibitors is similar to that of most current MMP inhibitors.7,10
Although some of these compounds entered the clinic, their development was eventually halted. Concerns were raised about the selectivity of this class of compounds versus human metalloproteases.11
Hydroxamic acid-based PDF inhibitors are likely to target other MPs because most of the binding energy for these compounds is derived from the chelation of the metal ion in the active site of MPs.7
Furthermore, actinonin has potent activity against various other MPs such as aminopeptidases M and N, leucine and alanyl aminopeptidases, enkephalin aminopeptidase, dipeptidyl aminopeptidase, enkephalinase A, and meprin α and β12,13
The lack of commercially available profiling reagents and services for MPs, as we encountered upon completion of our screening efforts for inhibitors of human peptide deformylase (HsPDF) (Antczak et al., manuscript in preparation), have prompted us to conceptually design a simple and generic assay. We envisioned that the lack of selectivity of actinonin could be turned into an advantage. We had previously designed a probe based on a derivative of actinonin, which allowed us to develop a fluorescence polarization (FP)–based competition assay for HsPDF.14
We hypothesized that the promiscuous activity of actinonin was probably not limited to its currently described targets, and therefore we reasoned that our probe might allow us access to a wider range of MPs. In this article, we describe a profiling platform that allows not only the identification of novel MPIs but also their selectivity profiling using this simple, generic, homogeneous, and cost-effective assay. We also demonstrate the proof of principle of this novel approach and discuss its implications for the prioritization of high-throughput screening (HTS) obtained hits for MPs.