Decoupling the Allosteric Activation of Ubiquitin Specific Protease 7 by Its Ubiquitin‐like Domains for the Development of Small Molecule Inhibitors

Ubiquitin specific protease 7, USP7/HAUSP, regulates cellular levels of E3 ligase‐MDM2 and tumor suppressor p53 by the deubiquitination and subsequent stabilization of MDM2. The up‐regulation of USP7 in many types of cancer make it an ideal target for the development of anti‐cancer therapeutics. It is well known that the catalytic domain of USP7 has 100‐fold less activity than that of the full length enzyme. Recent work by Fasen et. al. (2011) reported that the catalytic function of USP7 is activated through the C‐terminal HAUSP ubiquitin‐like (HUBL) domain interaction with the switching loop of the catalytic domain. Our studies, utilizing a fluorescence based kinetic assay, define the kinetic parameters that surround the coupling of the activation of the catalytic domain by the HUBL domains. A small molecule inhibitor only capable of inhibiting the full length enzyme was identified by high‐throughput screening of a 20,000 compound library at Purdue University. Analogues of this compound were synthesized and significant structure activity relationships were defined. By kinetic studies, we probed the mode of inhibition of the best analogues and identified the need of specific HUBL domains for the inhibition of the enzyme by these compounds. These compounds can therefore serve as molecular probes in decoupling the allosteric activation of the HUBL domains for further studies of the intra‐molecular interactions of USP7. Support or Funding Information This work is funded by a grant from the Walther Cancer Foundation