Rational design of bifunctional protein degrader drugs

双功能蛋白降解药物的合理设计

• 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
• 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

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性质和功能。的蛋白酶体降解 靶蛋白是Ub蛋白酶体系统（UPS）控制蛋白质含量的公认方法。 被称为Ub E3连接酶的酶通过在ε-连接蛋白之间形成异肽键将Ub结合到靶蛋白上。 靶蛋白赖氨酸的氨基和Ub的C-末端甘氨酸的羧酸酯。缀合 多个Ub在其7个赖氨酸（K）中的任一个处形成多聚Ub链，并且K6、K11、K27、K29、K33、K48和K63 具有各种作用的Ub链存在于所有组织中。最近，几个小组设计了小分子 其一端与E3连接酶结合，另一端与靶蛋白结合，在物理上促进蛋白质的泛素化。 目标蛋白，然后被降解。这种劫持连接酶来泛素化所需蛋白质的方法， 一种叫做PROTAC（PROteolysis Targeting Chimeras）的新药。基于PROTAC的方法， 治疗提供了几个优点：1）靶的选择性、催化降解; 2）将弱的 结合剂转化为选择性PROTAC药物; 3）降解过表达或突变的靶点;和4）最大 从有限的目标接触降级。到目前为止，cereblon和VHL连接酶结合剂已经是最常见的。 通常用作遍在化靶蛋白如核受体、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PROTAC的发展 然而，毒品。PROTAC分子的化学优化取决于合成的 化合物来指导用于产生药物候选分子的合成策略。目前可用的检测试剂盒 是劳动密集型的，不能足够快地向药物化学家提供结果-通常需要一周。 在第一阶段，开发了一种采用Ub连接酶cereblon和HDM 2的简便体外方法来筛选 潜在的PROTAC药物; PROTAC介导的所选蛋白质的泛素化在体外被重现， 这种方式模拟了体内观察到的PROTAC依赖性泛素化和这些蛋白质的降解， 实现第一阶段的目标。在第二阶段，将扩大这一方法的效用， 所有Ub连接酶家族的成员（cullin家族、RING指连接酶、Hect家族连接酶和SUMO连接酶）， 增加了PROTAC药物发现的生物化学和化学空间。要按比例放大PROTAC屏幕， 将建立基于微量滴定板的高通量方法来监测体外PROTAC药物发现， 生物化学和Ub质谱蛋白质组学将用于证明靶蛋白赖氨酸 在体外泛素化与体内PROTAC介导的靶蛋白降解相关。 基于微量滴定板的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。