Targeting ISG15 and USP18

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

USP18 is the only ISG15-specific protease identified to date although several DUBs have been reported to be cross-reactivity with ISG15 in vitro, such as USP2, USP5, USP14 and USP21. For development of a selective USP18 activity-based probe (ABP), we hypothesized that creating mutations in certain regions of ISG15 can optimize intermolecular contacts directing it towards recognition of USP18 over other deISGylases and provide selective USP18 ABP. We have identified residues in ISG15 that can be mutated through positional scanning analysis using both a combinatorial peptide library and in silico modeling. For example, our initial results showed that R153Agb and H90F mutations might enhance binding affinity and selectivity towards USP18 over other DUBs. We have generated these ISG15 mutants using a combination of solid-phase peptide chemistry and native chemical ligation strategies and measured their binding affinity towards USP18 in comparison to WT. With a covalent warhead and reporter tag (fluorophore or affinity handle) incorporated, we confirmed efficient labeling of USP18 by these mutants. More importantly, candidate mutants were unable to label USP5, indicating improved selectivity. With a successful preparation of ISG15-based probes, we will perform activity-based protein profiling (ABPP) of USP18 in different cancer cell lines and tumor samples of diverse origin. This study will inform us about the tumor types and states in which USP18 is highly activated and provide insights into the cellular context that USP18 inhibitor can have translational potential. We are also developing selective small molecule inhibitors of USP18. We have developed and optimized a biochemical assay that is suitable for high-throughput screening to identify catalytic inhibitors of USP18. After procuring high amount and quality enzymes and fluorogenic substrates, we've begun working with NCATS to screen inhibitors. Initial screening of protease inhibitor collection validated robustness and reproducibility of assay and identified some interesting molecules including this SJB compound known as a USP1 inhibitor and degrader. We are currently screening Genesis compound library that contains 125K compounds. From the first round of 45K compounds screening, we chose 128 compounds to follow-up at 11 pt dose response. A series of compounds showed promising low micromolar IC50 values. In parallel, we are conducting virtual ligand screening for non-covalent inhibitors. To evaluate the potential covalent targeting of USP18, we have developed an intact-protein mass spectrometry (MS) assay. Additionally, we are also employing small-molecule microarray (SMM) screening to identify small molecule binders to USP18. Once we have identified lead inhibitors, we will drive the mechanistic, structural, and biological evaluation of USP18 inhibitors through a concerted effort of independent and collaborative research. Specifically, my laboratory will carry out chemical optimization, biochemical, biophysical and cell biological analyses of these molecules. Lead molecules showing activity will be prioritized based on high potency, selectivity, and synthetic tractability. Active molecules will be validated in functional assays that probe protein ISGylation and IFN pathways. Once validated, probes will be used to define USP18's role in cancer development and its therapeutic potential.