Theme leaders: Huib Ovaa (NKI) and Hermen Overkleeft (LU)

Background

Chemical Biology aims at bringing chemical solutions to biological questions. The proteome can be subdivided in chemically related sub-proteomes that are distinguished by specific post-translational modification patterns or enzymatic activities. These distinguishing features can be capitalized upon by the development and application of techniques that are rooted in organic chemistry and/or analytical chemistry, and that enable their specific detection and/or modification. Some approaches have reached a mature state whereas others are in early development stages. Recurring difficulties are the extent and specificity with which sub-proteomes are selected or modified by means of organic chemistry, and the ensuing detection and the subsequent identification of these from complex proteomes by analytical and biochemical techniques. Chemical biology approaches provide useful means to detect proteins the identity of which is already known. However, its true potential lies in the assistance in unearthing unknown species, reducing bias.

In this theme we bring together a mix of chemical biology approaches that revolve around three specific subproteomes, namely kinases, phosphatases and proteasomes. The proposed studies include chemical biology approaches to monitor the enzyme families themselves, but also their substrates and products in physiological settings. The research is divided in ten separate but highly interrelated projects that are equally distributed over the five participating partners. The projects are a mix of academic projects and more advanced projects that were successfully initiated in NPC1 and that are now in a validation stage. With respect to the kinase research, we have found that protein kinase B (PKB) activity is upregulated by several bacterial strains, including m. tuberculosis, in their subcellular survival strategy. PKB thus forms a target for antibiotics development. PKB is a known and important target in oncology research and our lead PKB inhibitors may thus find application in two important therapeutic indications. We further identified by means of a kinome-wide RNAi knock-down screen a number of kinase activities that are part of the PKB signaling network and have therefore also therapeutic potential. We will bring to bear our advanced serine/threonine kinase peptide arrays as well as our kinase bait technology to profile kinase activities in infected tissue treated or not with inhibitors. We will transform our lead inhibitors into activity-based probes (ABPs) and match the results of the activity-based proteomics profiling (ABPP) experiments with the bait- and chip experiments. We will apply the same strategy to networks revolving around other kinases of interest, such as protein kinase A (PKA) in oncology. We have developed already extensive chemical proteomics tools to investigate PKA signaling within NPC1. The phosphatase project is planned to develop chemical biology approaches to study the factors involved in dephosphorylation. Together with kinases, phosphatases control phosphorylation events in cellular signaling, however the means to study these activities are scarce. We will initially focus on tyrosine phosphatases for which we will develop peptide arrays, inhibitors, baits and ABPs, and we will translate the results to serine/threonine phosphatases in a later stage. The proteasome project brings together our considerable array of chemical biology technology we have amassed over the years. Some fundamental questions are the heterogeneity of proteasome assemblies, the role of post-translational modification in proteasome functioning and degradation of the proteasome itself. We will validate our inhibitors and ABPs to monitor proteasome functioning in cancer, with possible directions for drug development as a result.

Approach

We have opted for an approach in which ten PhD students will work closely together on the three themes. Each PhD student will tackle an individual project that is part of the larger scheme, and each sub-theme (kinases, phosphatases and proteasomes) will be elaborated by three/four PhD students. Overlap between the sub-themes (for instance the bait/ABP development and the peptide array for both kinases and phosphatases) ensures close interaction between all PhD projects. The three sub-themes are selected on the basis of shared interests, for instance proteasome biology (Ovaa, Overkleeft, Neefjes, Heck), kinase biology (Neefjes, Overkleeft, Ruijtenbeek), kinase profiling (Neefjes, Ruijtenbeek, Liskamp, Heck) and phosphatase biology (Ovaa, den Hertog). The five partners together bring expertise in chemical biology (Ovaa, Overkleeft, Liskamp, Ruijtenbeek), organic synthesis (Overkleeft, Liskamp, Ovaa), cell biology (Neefjes) and analytical chemistry and affinity based mass spectrometry (Heck) and it is our intention to let the PhD students pursue parts of their research in the laboratory that is most suited for the specific task at hand. For instance, the building of libraries of inhibitors and baits that are based on a lead defined somewhere in the theme will be executed in the Overkleeft lab; advanced cell biology research will be hosted by Neefjes, cell based selectivity and potency profiling by Ruijtenbeek and Liskamp, and affinity profiling by Heck.

Deliverables

• Efficient selective proteasome sub-unit inhibitors and ABPs
• Efficient selective kinase (PKB and PKA and PKC) inhibitors and ABPs
• Efficient selective tyrosine phosphatase inhibitors
• Identification of novel targets of the inhibitors and their interactors
• Novel peptide array based kinase and phosphatase assays
• Novel siRNA based kinase and phosphatase assays
• Identification of target kinases and phosphatases in oncology and infectious diseases