Project No.2218

Primary Supervisor

Prof Steve Goldup- University of Southampton

Co-Supervisor(s)

Dr Matthias Baud – University of Southampton

Prof Michelle Garrett – University of Kent

Summary

Background and significance:

Recent years have seen the emergence of new classes of chemical probes where two small molecule chemical probes are “linked” together by a chemical linker, to enforce a new interaction between two Proteins Of Interest (POIs) and redirect them towards a particular cellular fate (notably proteasomal degradation/knock-down by PROTAC probes acting via the UPS) [Chem. Rev. 2017, 11269]. These hetero-bifunctional probes have generated huge interest and excitement and significant current research is directed towards applying them i) to knock-down “undruggable” proteins and their signalling pathways [Cell Research 2016, 484]; and ii) to optimise their properties for use in vivo to address unmet therapeutic needs in areas such as cancer and neurodegenerative conditions. However, modulation of key properties such as membrane permeability, aqueous solubility, metabolic stability and biodistribution have proven challenging [Explor. Target Antitumor. Ther. 2020, 273-312, most cited article of the journal]. Proposal: In this proof-of-concept study, we will address this challenge by threading the linker PROTAC probes through a stable molecular ring to generate a structure called a “rotaxane”. Goldup (PI) has developed methods to prepare such rotaxanes using “click” chemistry [J. Am. Chem. Soc. 2016, 16329], and demonstrated that threading DNA through a molecular ring effectively modulates its properties [J. Am. Chem. Soc. 2020, 5985]. In simple terms, our hypothesis is that threading the linker of PROTAC probes through a molecular ring will change its biological properties in a modular and straightforward manner. This will allow the activity of PROTACs to be tuned quickly and effectively for the enhanced cellular knock-down of challenging POIs, and perhaps enhanced efficacy in full organisms going forward. Objectives: 1. Design and synthesise rotaxane-PROTACs (“matched pairs”), varying the structure of the threaded macrocycle and characterise their physico-chemical properties in biophysical assays. 2. Evaluate rotaxane-PROTACs in vitro versus the parent non-rotaxane probes using a palette of molecular/cell biology techniques. 3. Use optimised rotaxane-PROTACs to interrogate the CHK1 kinase pathway -a critical regulator of the DNA damage response which plays multiple pivotal functions in human physiology and cancer, in order to demonstrate the power of the rotaxane-PROTAC approach.