Developing novel biophysical and cellular assays to study small-molecule mediated protein ubiquitination and degradation

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Bifunctional degrader molecules also known as Proteolysis Targeting Chimeras (PROTACs) and the mechanistically related monovalent degraders (for example auxin, lenalidomide and indisulam) work by co-opting an E3 ubiquitin ligase to act upon a neo-substrate protein. Formation of a ternary complex species between the E3 ligase, the degrader molecule and the target protein leads to the latter being ubiquitinated and subsequently degraded by the proteasome. Because of their different mechanism of action, and the advantages of targeted protein degradation as a pharmacological outcome, degraders are emerging as a new paradigm modality of chemical tools to probe biology and molecular therapeutics.The Ciulli Lab's biophysical and structural studies of PROTAC ternary complexes have illuminated important features to the PROTAC mode of action, for example the relevance of inducing neo-protein-protein interactions that contribute to forming cooperative and stable ternary complexes [1-2]. The sub-stoichiometric nature of the PROTAC mode of action as well as the dynamic and complex equilibria for ternary complex formation require the development of bespoke assays in various modalities to better understand the nuances of the mechanism for different combination of E3 : PROTAC : target [3]. Furthermore, ternary complex formation, albeit required and so necessary for the mode of action, alone is notsufficient because effective productive target ubiquitination needs to occur to trigger rapid protein degradation. Monitoring the molecular events downstream of ternary complex formation is therefore important to gain a full picture of the process.This project aims to develop a suite of biophysical and cellular assays to elucidate and dissect the individual steps of the mechanism of action of PROTACs and molecular glues, namely: 1) binary target engagement; 2) ternary complex formation equilibria and kinetics;3) protein ubiquitination; 4) proteasomal recruitment and degradation. The project is designed as highly interdisciplinary, drawing on complementary expertise from both the Ciulli lab (expert in PROTAC mechanisms) and the Hay lab (expert in protein ubiquitination mechanisms). The student will receive outstanding training in multidisciplinary areas incutting-edge research in fundamental and translational chemical and structural biology from both labs.The project will be developed as iCASE partnership in collaboration with Tocris, because of their strong interest in the development and application of PROTACs and related technologies. Given the strong relationships established by the main applicant with the company in the area of development and commercialization of chemical probes and PROTACs that have originated from the Ciulli Lab, we anticipate strong interest andcommitment from the company involved. A letter of support is included in attachment.References1. Maniaci C, Ciulli A. Curr Opin Chem Biol 2019; 52:145-56.2. Roy M et al. ACS Chem Biol 2019; 14:361-8.3. Zoppi V et al. J Med Chem 2019; 62:699-726.