|Ph.D Student||Azulay Shimrit|
|Subject||Novel Chemical Approaches for Studying and Targeting|
|Department||Department of Chemistry||Supervisor||Professor Ashraf Brik|
|Full Thesis text|
Deubiquitinases (DUBs) are proteolytic enzymes that counteract ubiquitination, which is one of the highly important post-translation modification and is associated with different cellular signaling and, hence, in several diseases. Targeting DUBs as a therapeutic approach for various diseases, such as for cancer treatment, is currently an important goal both in academia and industry. In order to study DUB activities and to enable their targeting with small molecules and peptides, a very efficient assay is required. Along these lines, and as part of the thesis goals, a rapid and efficient synthetic method was developed to improve our previously developed quenching pair assay for USP2 and USP7.
Using our developed assay, we screened a focused library based on quinones against USP2, which showed a strong preference for an ortho- vs. para-quinone scaffold. Further mechanistic studies of the selected compounds revealed selective oxidation of the active site Cys, to the irreversible sulfinic acid, via reactive oxygen species (ROS). Such a discovery is the first of its kind where small molecules that are capable of generating ROS can be used to inhibit DUBs. Testing these compounds with a DU-145 prostate cancer cell line exhibited an increased level of apoptosis, due to the role of USP2 in the protection of cancer cells from undergoing apoptosis. Interestingly, one of our best compounds was b-lapachone?a natural product currently in clinical trials for treating different kinds of cancer. Our results suggest a possible new mechanism for b-lapachone toxicity for various cancer types. Cellular studies on the effect of these inhibitors on USP2 substrates have revealed a decrease in Cyclin D in a dose- and time-dependent manner.
Combining electrochemistry, synthesis of various quinone analogues, and biochemical analyses, a rational understanding of the ability of small molecules to generate ROS and inhibit USP2 was achieved. Interestingly, a clear preference of ortho- vs. para- isomers was observed suggesting that the reduction of molecular oxygen to generate ROS is efficiently catalyzed by ortho- compared to para-isomers. Substitutions of the ortho-quinone molecules were also tested for their influence on generating ROS. Notably, a narrow range of redox potential was found to be efficient in the catalysis of oxygen reduction that finally results in USP2 inhibition.
Another main goal of this thesis was the search for novel inhibitors other than ROS- generating molecules. Optimization of our substrate and its large scale synthesis enabled us to perform high throughput screening of ~90,000 compounds, which revealed new hits for USP2 inhibition. These new hits appear to operate via a non-covalent mode of inhibition, and are currently undergoing further modifications to optimize their potency.
Peptide-based inhibitors as a new scaffold to inhibit DUBs were also investigated. Truncation of ubiquitin, based on its secondary structure motifs, was performed to yield five peptides in the range of 10-20 residues. Interestingly, mutations in the N-terminal peptide composed from 17 amino acids, which is known to adopt a stable secondary structure in solution, led to USP7 inhibition. Currently, this peptide is also being optimized for better potency using peptide synthesis and computational tools.