Abstract:
USP7 (ubiquitin-specific protease 7), also known as HAUSP (herpes-associated ubiquitin-specific protease), is one of over 100 deubiquitinases that removes ubiquitin from certain proteins, thereby protecting the protein from degradation. Elevated USP7 levels in the human body are detected in some types of tumours, such as prostate carcinomas, neuroblastomas and glioblastomas, and are associated with aggressive disease progression.
This work focused primarily on the covalent addressing of catalytic cysteine 223 of USP7. Firstly, fragments from a covalent fragment library (CovLib) were examined, including their structure-activity relationship (SAR) fragments. Differential scanning fluorimetry (DSF) was used as primary screening method. The detection of a possible covalent modification of USP7 was then carried out using intact protein mass spectrometry with additional control mutants. This screen yielded several reactive fragments, mostly with an SNAr warhead. They lowered the melting temperature of USP7 and modified it covalently several times. Interest in larger, less reactive fragments increased, leading to a further screen of a covalent library originally synthesised for kinases. Seven fragments modified covalently USP7 from this library. The exact position of the binding of a fragment of the kinase inhibitor library (GCL36) with USP7 could be shown by a crystal structure. GCL36 was most likely the only compound, compared to the published structures in the protein database (PDB), that was oriented towards the catalytic triad and not towards the ubiquitin tunnel. This crystal structure served as a starting point for the synthesis of a SAR series of the GCL36 fragment.
The novel binding mode of the synthesised fragments still needs to be confirmed by protein crystallography. Future studies should focus on the identification, design and synthesis of high-affinity compounds inhibiting USP7 in order to achieve progress in tumour therapy.
USP7