Rational design of bifunctional protein degrader drugs

双功能蛋白降解药物的合理设计

基本信息

批准号：
10259221
负责人：
Karteek Kadimisetty
金额：
$ 100.42万
依托单位：
LIFESENSORS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2023-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10259221
关键词：
Autophagocytosis Binding Biochemical Biological Assay Biology Biotechnology C-terminal Cell Line Cell physiology Cells Chemicals Clinic Cullin Proteins Development Enzymes Evaluation Excision Family Genes Glycine Goals Human Genome Immunoblotting In Vitro Lead Ligands Ligase Luciferases Lysine MDM2 gene Mediating Metabolism Methodology Methods Monitor Morphologic artifacts Neurons Nuclear Receptors Pharmaceutical Preparations Phase Phosphotransferases Play Polyubiquitin Process Progress Reports Promega Property Protac Proteins Proteomics Reporter Research Ring Finger Domain Role Specificity Structure-Activity Relationship System Testing Therapeutic Time Tissues Translating Ubiquitin Ubiquitination alpha synuclein amino group base carboxylate chimera drug clinical development commercialization design drug candidate drug development drug discovery in vivo live cell imaging member multicatalytic endopeptidase complex mutant novel drug class novel therapeutic intervention overexpression protein degradation protein function protein transport rapid test receptor recycling reconstitution scale up screening small molecule success tau Proteins transcription factor ubiquitin ligase ubiquitin-protein ligase

项目摘要

Ubiquitin (Ub) tags regulate multiple properties and functions of proteins in cells. Proteasomal degradation of target proteins is a well-established means whereby the Ub proteasome system (UPS) controls protein content. Enzymes called Ub E3 ligases conjugate Ub to target proteins by forming an isopeptide bond between the ε- amino group of the target protein lysine and the carboxylate of the C-terminal glycine of Ub. Conjugation of multiple Ubs forms poly-Ub chains at any of its seven lysines (K), and K6, K11, K27, K29, K33, K48, and K63 Ub chains having various roles are present in all tissues. Recently, several groups designed small molecules that bind to an E3 ligase at one end and a target protein at the other, physically facilitating ubiquitylation of the target protein, which is then degraded. This hijacking of a ligase to ubiquitylate a desired protein has launched a new class of drug called PROTACs (PROteolysis TArgeting Chimeras). PROTAC-based approaches for therapeutics offer several advantages: 1) selective, catalytic degradation of the target; 2) conversion of weak binders into selective PROTAC drugs; 3) degradation of overexpressed or mutant targets; and 4) maximal degradation from limited target engagement. To date, cereblon and VHL ligase binders have been most commonly used as vehicles to ubiquitylate target proteins such as nuclear receptors, kinases, transcription factors, and neuronal proteins tau and α-synuclein. A major problem has hindered development of new PROTAC drugs, however. Chemical optimization of PROTAC molecules depends on rapid evaluation of synthesized compounds to guide the synthetic strategy for producing drug candidate molecules. Assays currently available are labor intensive and do not provide results to the medicinal chemists fast enough – often, a week is required. In Phase I, a facile in vitro method employing Ub ligases cereblon and HDM2 was developed to screen for potential PROTAC drugs; PROTAC-mediated ubiquitylation of selected proteins was recapitulated in vitro in a way that mimics observed PROTAC-dependent ubiquitination and degradation of these proteins in vivo, achieving the aims of Phase I. In phase II, the utility of this method will be expanded to include representative members of all Ub ligase families (cullin families, RING finger ligases, Hect family ligases, and SUMO ligase), increasing the biochemical and chemical space for PROTAC drug discovery. To scale up PROTAC screens, a microtiter plate-based, high throughput method will be established to monitor in vitro PROTAC drug discovery, and biochemical and Ub mass spec proteomics will be employed to demonstrate that target protein lysines ubiquitylated in vitro are correlated with in vivo PROTAC mediated degradation of target proteins. Commercialization of the microtiter plate based PROTAC system will have a major impact on academic research as well as PROTAC drug discovery.

泛素（UB）标签调节细胞中蛋白质的多种特性和功能。蛋白酶体降解靶蛋白是一种良好的手段，UB蛋白质组系统（UPS）控制蛋白质含量。称为UB E3连接酶的酶通过在ε-之间形成异肽键来靶向蛋白质。靶蛋白赖氨酸的氨基和Ub的C末端甘氨酸的羧酸盐。共轭多个瑞银在其七个赖氨酸（K）和K6，K11，K27，K29，K33，K48和K63中的多个链条链中的多个链条形成poly-ub链在所有组织中都存在具有各种作用的UB链。最近，几个小组设计了小分子在一端与E3连接酶结合，另一端结合靶蛋白，在物理上支持泛素化。靶蛋白，然后降解。这种连接酶的劫持以泛素化酸盐已发射一种称为Protacs的新药物（靶向嵌合体的蛋白水解）。基于Protac的方法治疗药具有多种优势：1）靶标的选择性，催化降解； 2）弱的转换粘合剂进入选择性protac药物； 3）过表达或突变靶标的降解； 4）最大从有限的目标参与中退化。迄今为止，Cereblon和VHL连接酶粘合剂最多通常用作泛素酯靶蛋白的车辆，例如核接收器，激酶，转录因子和神经元蛋白Tau和α-突触核蛋白。一个主要问题阻碍了新的Protac的发展但是，药物。 Protac分子的化学优化取决于合成的快速评估化合物指导生产候选药物分子的合成策略。当前可用的测定是劳动密集型的，并且不能足够快地为药物化学家提供结果 - 通常需要一周。在第一阶段，开发了一种使用UB连接酶Cereblon和HDM2的体外方法，以筛选潜在的protac药物；在体外概括了Protac介导的选定蛋白的泛素化。模仿在体内这些蛋白质的Protac依赖性泛素化和降解的方式，实现第一阶段的目标。在第二阶段，该方法的实用性将扩展到包括代表性所有UB连接酶家族的成员（Cullin家族，环手指连接酶，Hect家族连接酶和SUMO连接酶），增加生化和化学空间以发现Protac药物。为了扩展Protac屏幕，将建立基于微滴定板的高吞吐量方法，以监测体外Protac药物发现，并且将聘请生化和UB质量规格蛋白质组学以证明目标蛋白质歌词在体外的泛素化与靶蛋白的体内介导的降解相关。基于微滴定板的Protac系统的商业化将对学术研究产生重大影响以及Protac药物发现。