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Chemical Proteomics Applied to Target Identification and Drug Discovery
Steven H.L. Verhelst Matthew Bogyo
Stanford University School of Medicine, Stanford, CA, USA
BioTechniques, Vol. 38, No. 2, February 2005, pp. 175–177
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In the past decade, DNA sequencing of multiple organisms has provided us with our first look at complete lists of protein products expressed from a whole genome. The field of proteomics is now challenged with the task of elucidating the cellular functions of these proteins in both normal and pathological processes (1,2,3). Several proteomic techniques, such as 2-dimensional gel-electrophoresis (4) and isotope-coded affinity-tagging (5), provide a means to globally monitor changes in protein expression levels. However, the abundance of a protein is not necessarily directly coupled to its activity. Most enzymes are mediators of biochemical transformations and are therefore highly posttranslationally regulated. Many are synthesized as zymogens, which are tightly regulated through enzymatic activation. Moreover, the activity of enzymes is altered by changes in pH and the presence of inhibitors. Thus, methods that allow direct quantification of protein activities rather than simple abundance are required to uncover distinct protein functions in physiological and pathological events.

A new research field has been created to begin to address protein function at the level of regulation of enzyme activity. This new area has been given the name chemical proteomics, or activity-based proteomics, and makes use of small molecules that can covalently attach to catalytic residues in an enzyme active site (6,7,8,9,10,11). The selectivity of the chemically reactive group allows specific proteins or protein subsets to be tagged, purified, and analyzed. As a result, this technique is able to identify novel enzymatic proteins and has the potential to accelerate the discovery of new drug targets.

Activity-Based Probe Design

The small molecules used in chemical proteomics, so-called activity-based probes (ABPs), generally consist of three basic elements ((Figure 1)). The warhead consists of a reactive functional group that is able to covalently attach to the active site of an enzyme, while the tag can be used for identification and purification of modified enzymes. The linker connects these two elements, provides selective binding interactions, and prevents steric congestion.

Figure 1.

The anatomy of an activity-based probe (ABP). Examples of two probes that use different reactive warheads, different linkers, and the same biotin tag. These probes target cysteine proteases (DCG-04) and serine hydrolases (FP-Biotin).

The warhead is perhaps to most critical component of an ABP. On one hand, it should be reactive enough to modify somewhat latent nucleophiles. However, its chemical reactivity must not lead to nonspecific modification of other abundant molecules within the proteome. The majority of ABPs use electrophilic groups, often derived from irreversible enzyme inhibitors (12) that are able to form a covalent bond with a nucleophile located in the enzyme active site. Two examples include the cysteine protease probe DCG-04 (13) and the serine hydrolase probe FP-biotin (14) ((Figure 1)). The former is derived from the natural product E-64, a broad-spectrum cysteine protease inhibitor. The warhead of the latter resembles the general serine protease inhibitor diisopropyl fluorophosphate.

Besides being a simple connection between warhead and tag, the linker can influence the specificity of the ABP. In this respect, polypeptides are often used as linkers due to their ease of preparation and compatibility with solid phase chemistry. In addition, many enzymes have protein substrates and therefore bind probes with peptide character. Furthermore, information regarding substrate specificity of a given enzyme can be used to design probes that display selectivity for distinct enzyme targets. For instance, the leucine residue in DCG-04, situated next to the epoxide electrophile, directs its selectivity towards the papain family of cysteine proteases, all of which possess hydrophobic P2 pockets. On the other hand, FP-biotin was designed as a general serine hydrolase probe. Therefore, a simple alkyl spacer was chosen to connect the fluorophosphonate to the reporter biotin-group. Consequently, this probe is not limited to a single subclass, but exhibits reactivity towards a broad range of serine hydrolases.

Besides the above described approach using electrophilic warheads, two other strategies have been developed for the design of ABPs. Both of these methods rely on the generation of a highly reactive electrophile in the active site leading to alkylation of a nearby nucleophile on the target. The first class of affinity-based labeling probes contains photolabile groups that can be converted into a highly reactive intermediate after irradiation with UV light (15,16). This class of compounds has been used to label metallo-proteases that do not use a side-chain nucleophile to directly attack a protein substrate. The second class consists of a masked electrophile that becomes activated upon cleavage by the enzyme (17,18,19,20). These reagents serve as bona fide enzyme substrates and only become converted into reactive probes after turnover. While this method seems to have promise, it is not yet clear if product release is too rapid, thereby leading to highly nonspecific labeling of other proteins.

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