Validation of replication licensing as a therapeutic anti-cancer target via a novel VHL-based PROTAC inducible degron technology

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In human cells, the initiation of DNA replication is regulated via the licensing checkpoint which is often abrogated in cancer cells. Previous research suggests that cancer cells deficient in the licensing checkpoint are vulnerable to licensing inhibition. However, this hypothesis requires more rigorous validation. Target validation via small molecule inhibitors comes with many problems and novel technologies are in demand. This research proposes to develop and apply a novel inducible degron based on PROTACs (proteolysis targeting chimaeras) selective for an unnatural bromodomain to the replication licensing pathway to expedite its target validation as a therapeutic anti-cancer target. Development of this novel degron technology will implement biophysical (ITC, FRET), biochemical (Western Blot, pull-down), and genetic (CRISPR cas9) approaches.Key Words and Skills:1. Replication Liscensing 2. Cancer3. Target Validation 4. Novel Degron technology 5. PROTACs Key Skills: Western Blotting ITC CRISPR Cas9 FRET Microscopy FACS Tissue Culture In vitro Liscensing assay Pull down Antibody validation Questions:1. Explain interdisciplinary interface: Development of this novel degron technology will require analysis of the interaction between the unnatural bromodomain and the PROTAC compounds that is dependant of biophysical techniques such as ITC, FRET, AlphaLISA and X-ray Crystallography. This project is also reliant on compound synthesis which will be dependant on information produced from data identified during this project. 2. Does project require significant amount of quantitative skills? NO (delete as appropriate)3. Does project require significant amount of whole organism physiology skills? NO (delete as appropriate)