Summary
Activity-based protein profiling (ABPP) techniques that make use of organic chemical baits provide an attractive strategy for the assessment of the expression levels and functioning of enzyme families in the context of the proteome as a whole. One of the main attractions of ABPP strategies that make use of so-called activity-based probes is that they report on enzyme activity levels, rather than mere expression levels. On the downside, there is as yet no general set of rules that guide the design of ABPPs for a given enzyme, or enzyme family. Not surprisingly, activity-based protein profiling has matured most for those enzymes that are not too restrictive where it comes to their substrates, and whose mode of action in catalysis directly involves a specific amino acid residue present in their active site. Such enzymes can often be irreversibly modified by means of a molecule that contains an electrophilic trap (often referred to as ‘warhead’) and an affinity-identification tag (biotin, matrix, fluorophore). In this fashion probes targeting several protease families (the proteasome, ubiquitin isopeptidases, cysteine proteases) were developed with considerable success. Some enzyme families, such as exoglycosidases, however are rather more restrictive with respect to the nature of the substrate mimic and affinity/identification tags for these enzymes need be installed in a two-step bioorthogonal fashion. Other enzyme families, such as the matrix metalloproteinases, exert a mechanism of action that involve a water molecule as the reactive species and these cannot be modified irreversible by means of an electrophilic trap. Here, activity-based probes containing a photoactivatable group come to the rescue, but the efficiency of these is often rather limited. The enzyme family that is subject of this project, kinases, suffers from both disadvantages and it is therefore not surprising that no general ABPP tool to monitor large subsets of this important and large subproteome is available yet. We intend to develop just such a set of tools. The proposed research is based on the one hand on the success we had in the past in the development of a set of serine/threonine kinase inhibitors biased to some extend to PKB/Akt1, and on the other hand on our expertise in the development of general activity-based protein profiling tools that incorporate one or more of the above features.