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

双功能降解物分子也被称为针对嵌合体的蛋白水解酶(PROTACs)和与机制相关的单价降解物(例如生长素、来那度胺和吲哚)，其工作原理是通过增选E3泛素连接酶作用于新底物蛋白。在E3连接酶、降解物分子和靶蛋白之间形成一个三元复合体，导致后者被泛素化，随后被蛋白酶体降解。由于它们不同的作用机制，以及靶向蛋白质降解作为药理结果的优势，降解剂正在成为探索生物学和分子治疗的一种新的化学工具的范例模式。Ciulli实验室对PROTAC三元复合体的生物物理和结构研究揭示了PROTAC作用模式的重要特征，例如，诱导新的蛋白质-蛋白质相互作用有助于形成合作和稳定的三元复合体[1-2]。PROTAC作用模式的亚化学计量性质以及三元络合物形成的动态和复杂平衡要求以各种方式开发定制的分析方法，以更好地了解E3：PROTAC：TARGET不同组合的机制的细微差别[3]。此外，尽管三元复合体的形成是作用模式所必需的，但仅有三元复合体的形成是不够的，因为需要发生有效的生产性靶标泛素化才能引发蛋白质的快速降解。因此，监测三元复合体形成过程下游的分子事件对于全面了解这一过程非常重要。本项目旨在开发一套生物物理和细胞分析来阐明和剖析PROTACs和分子胶作用机制的各个步骤，即：1)二元靶标结合；2)三元复合体形成平衡和动力学；3)蛋白质泛素化；4)蛋白酶体招募和降解。该项目设计为高度跨学科，利用Ciulli实验室(PROTAC机制专家)和Hay实验室(蛋白质泛素化机制专家)的互补专业知识。学生将从这两个实验室接受在基础和转化化学和结构生物学的尖端研究方面的多学科领域的杰出培训。由于他们对PROTAC和相关技术的开发和应用有着浓厚的兴趣，该项目将作为iCASE与托里斯的合作伙伴关系而发展。鉴于主申请人与该公司在化学探头和源自Ciulli实验室的PROTAC的开发和商业化领域建立了牢固的关系，我们预计相关公司将表现出浓厚的兴趣和承诺。附件中包括一封支持信。参考1。Maniaci C，Ciulli A.Curr Opin Chem Biol 2019；52：145-56.2。Roy M等人。《美国化学生物》2019；14：361-8.3。Zoppi V等人。J Med Chem 2019；62：699-726。

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.