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

双功能降解剂分子也称为蛋白水解靶向嵌合体（PROTAC）和机械相关的单价降解剂（例如生长素、来那度胺和indisulam）通过共同选择E3泛素连接酶作用于新底物蛋白而起作用。在E3连接酶、降解分子和靶蛋白之间形成三元复合物物种导致后者被泛素化并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学结果的优势，降解剂正在成为探测生物学和分子治疗学的化学工具的新范式。Ciulli Lab对PROTAC三元复合物的生物物理和结构研究阐明了PROTAC作用模式的重要特征，例如，诱导新蛋白质-蛋白质相互作用的相关性，其有助于形成协同和稳定的三元复合物[1-2]。PROTAC作用模式的亚化学计量性质以及三元复合物形成的动态和复合物平衡要求开发各种模式的定制测定，以更好地了解E3：PROTAC：靶标不同组合的机制的细微差别[3]。此外，三元复合物的形成，虽然是必需的，也是作用方式所必需的，但单独是不够的，因为有效的生产性靶泛素化需要发生以触发快速的蛋白质降解。本项目旨在开发一套生物物理和细胞分析方法，以阐明和剖析PROTAC和分子胶作用机制的各个步骤，即：1）二元靶点接合; 2）三元复合物形成平衡和动力学;3）蛋白质泛素化; 4）蛋白酶体的募集和降解。该项目被设计为高度跨学科的，借鉴了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。学生将在两个实验室接受多学科领域的优秀培训，这些领域是基础和转化化学和结构生物学的前沿研究。该项目将与Tocris合作开发为iCASE合作伙伴关系，因为他们对PROTAC和相关技术的开发和应用非常感兴趣。鉴于主要申请人与公司在开发和商业化化学探针和源于Ciulli实验室的PROTAC领域建立了牢固的关系，我们预计相关公司会表现出强烈的兴趣和承诺。支持信见附件。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.

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.