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

在人类细胞中，DNA复制的启动是通过许可检查点来调节的，而在癌细胞中，许可检查点通常是被废除的。先前的研究表明，缺乏许可检查点的癌细胞很容易受到许可抑制的影响。然而，这一假设需要更严格的验证。通过小分子抑制剂进行靶向验证伴随着许多问题，需要新的技术。本研究建议开发一种新的基于PROTACs(蛋白水解靶向嵌合体)的可诱导降解基因，并将其选择性地用于复制许可途径，以加快其作为治疗抗癌靶点的有效性。这项新的降解技术的开发将实现生物物理(ITC，FRET)、生化(Western Blot，下拉)和遗传(CRISPR Cas9)方法。关键词和技能：1.复制许可2.癌症3.靶标验证4.新型Degron技术5.PROTACs关键技能：Western blotting ITC CRISPR Cas9 FRET显微镜FACS组织培养体外糖化试验下拉抗体验证问题：1.解释跨学科接口：这项新型Degron技术的开发将需要分析非天然溴域与PROTAC化合物之间的相互作用，这依赖于ITC、FRET、AlphaLISA和X射线结晶学等生物物理技术。该项目还依赖于化合物合成，而化合物合成将依赖于该项目期间确定的数据所产生的信息。2.项目是否需要大量的量化技能？否(适当删除)3.项目是否需要大量的整体生理技能？否(适当删除)

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)