| Deubiquitinating enzymes (DUBs) are important regulators of protein degradation, -trafficking, DNA repair and epigenetic events. DUBs cleave ubiquitin (Ub), a proteinaceous post translational modifier from Ub-modified proteins. They can trim and disassemble ubiquitin polymers, releasing protein substrates from intended fates. A large fraction of the more than 100 DUBs encoded in the human genome is known to play a crucial role in the pathology of human disease, including cancer. DUB inhibitors, including ones of broadest possible specificity, are in great demand as DUB action complicates research aiming to study Ub modification. Cell lysis leads to a burst of DUB activity severely hampering the isolation and study of Ub-modified proteins. In addition, a large redundancy exists within the DUB family and thus systematic RNAi-mediated knockdown, although proven useful in this research area, is not always the tool of choice as knockdown may be compensated for. In analogy, studying the phosphorylation status of proteins would be unthinkable without the availability of the pan-phospatase inhibitor pervanadate. Such a tool is lacking in the ubiquitin field and it is the goal of this proposal to deliver it. Through data-mining pubchem, literature and patents we have identified and tested a panel of candidate DUB inhibitors. Five had high structural homology with IC50 values in the low μM range in independent assays; two of which inhibited all DUBs tested. Resynthesis of hits and synthesis of analogs led to active inhibition as well. All these structures are easily accessed by synthesis and one of them (indirubin U17, Table 1) is a known GSK3/CDK inhibitor and DUB inhibition may thus well contribute to some of the actions of this agent. Here, I propose to chemically evolve the identified structures in order to obtain potent cell permeable pan-DUB inhibitors as general tools for biomedical research. DUBs are known to undergo significant structural changes upon substrate binding, realigning their catalytic residues. It is at present not clear how to specifically inhibit the members of this family with a small molecule while a large homology exists within their catalytic domains. Detailed information on inhibitory structures will catalyze further research in this area as many consider this class of enzymes ?undruggable?. Cell-based structure-activity relations will guide the further development of these inhibitors and investigational tools based upon them. We will attempt to obtain structural information on DUB inhibitory motifs, while evolving molecules guided by activity in cell based assays scoring for proteasome activity, p53 stability and NFB translocation as I prefer this over in vitro approaches: specific DUB inhibition is likely hard to obtain, while the impact of very specific DUB inhibitors can be questioned. In analogy to kinase inhibitors, promiscuity often leads to better results and I deem cell-based approaches more useful to evolve structures, simultaneous selecting for favourable properties such as permeability, stability etc. In addition, many DUBs require an interacting partner for activity,1 favouring cell-based assays. The binding mode of these inhibitors will be studied independently as part of a related X-ray project (collaboration Sixma lab, NKI). Overall, the molecules and insights resulting from this research will not only open options for the future development of inhibitors of this notoriously difficult class of enzymes, but also allow the study of the ubiquitinated proteome and DUB-dependent biochemistry. |
