Technology Overview

ActivX's core technology is based on the work of co-founder Benjamin Cravatt, Ph.D., and his team at The Scripps Research Institute, which initially focused on developing tools to track the activity of the serine hydrolase super-family of enzymes. By coupling a classical serine hydrolase affinity label (flurophosphonate ester) to a detectable tag (biotin), a sensitive and versatile molecule was created that allowed for comprehensive profiling of serine hydrolase enzymes directly in complex biological mixtures. Because the probe was reactive only with catalytically active serine hydrolase enzymes, and not their inactive zymogens or inhibited forms, the approach was termed Activity Based Protein Profiling, or ABPP (Figure 1). Based on this initial success, Dr. Cravatt and others founded ActivX biosciences with the aim of expanding the scope of activity based probes (ABPs) to provide a powerful set of tools for drug discovery and development.

The basic principles behind ABPP are rooted in traditional affinity labeling methodology. ABPs are designed to target, or recognize, a specific feature of a protein/enzyme so that a covalent bond is formed between the probe and a specific amino acid side chain on the protein, typically in the active site. Historically, however, affinity labels either lacked sufficient selectivity to perform effectively in complex protein mixtures (i.e. proteomes), or were designed to specifically target a single protein. Each probe for ABPP on the other hand is carefully designed to target a conserved active site feature of a major enzyme class. As a result, a single ABP will theoretically react specifically with the active site of all members of the target enzyme family. This balance between probe specificity, to permit use of the probes in crude biological protein mixtures, and probe generality, to enable the comprehensive interrogation of a particular enzyme class, is the key to ABPP.

ABPs all share basic structural features including: a reactive group that can generate a stable, covalent bond with the target protein; a reporter group to enable detection and/or isolation of probe/protein reaction products; and a linker to connect the reactive group to the reporter group (Figure 2). Many probes also contain a recognition element that provides non-covalent binding energy to direct the probe to the enzyme active site and properly position the reactive group. A good example of a recognition group is seen on the ActivX kinase/ATPase probe which is built around the core structure of ATP (see Protein Kinase Probe page).

A simple, illustrative example of the power of ABPP is shown in Figure 3. In this experiment, rat liver membranes were treated first with an inhibitor of a serine hydrolase (DPPIV) and then with a serine hydrolase ABP tagged with a fluorophore (TAMRA). After labeling, the sample was loaded on an SDS-PAGE gel for electrophoresis, then scanned using a fluorescence flatbed scanner. Many bands can be seen, all of which represent serine hydrolase enzymes active in rat liver membranes. The profile of these enzymes is distinctly different than the overall protein pattern of the proteome observed by protein staining (left panel). In the presence of the DPPIV inhibitor, the probe is unable to access the DPPIV active site, and thus the DPPIV band, at ~100 kDa, is absent in this lane. This simple example highlights two important properties of ABPP. First, ABPP provides a fractionation method for proteomic analysis based on protein function. This focuses the analysis on enzymes of interest and greatly increases the sensitivity of the analysis relative to abundance based proteomics methods. Second, ABPP provides an efficient means of determining the selectivity of small molecule inhibitors (and drug candidates) against a broad range of related enzymes. Drug selectivity is key to avoiding toxicity and unwanted side effects. In many cases, enzymes covered by ActivX probes have no other assays available and thus cannot be screened by other methods. Additionally, the ABPP method allows for screening of all probe reactive enzymes in their native environment, avoiding potential artifacts related to recombinant protein expression.

For more than five years, ActivX scientists have been perfecting the art of creating activity based probes. Probes have been developed for targeting serine hydrolases/proteases, protein kinases, ATPases and other nucleotide binding proteins, cysteine proteases including cathepsins and caspases, oxidoreductases, and more. In addition, ActivX has developed a suite of powerful platforms for analyzing ABP labeled samples. More information on these analysis platforms and available probes, as well as examples of the types of data generated can be found by following the appropriate links above.