Development of a Tumor-Specific PROTAC

肿瘤特异性 PROTAC 的开发

• 批准号: 9752229
• 负责人: Michael Joseph Bond
• 金额: $ 4.5万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9752229
• 关键词: 26S proteasome; 5' -AMP-activated protein kinase; Address; Affinity; Antigens; Binding; Biological Assay; Biophysics; C-terminal; Calorimetry; Cancer Patient; Cancerous; Cell Culture Techniques; Cells; Chemistry; Chimera organism; Complex; Development; Dimerization; Double Minutes; Drug resistance; Effectiveness; Elements; Epidermal Growth Factor Receptor; Event; Flow Cytometry; Fluorescence; Fluorescence Polarization; Future; Gene Expression; Gene Family; Genes; Germ Lines; Growth; Homologous Gene; Lead; Libraries; Ligand Binding; Ligands; Malignant Neoplasms; Measures; Monitor; Mus; Mutation; National Cancer Institute; Non-Small-Cell Lung Carcinoma; Oncogenic; Oncoproteins; Peptides; Pharmacology; Phase; Protein Family; Protein Kinase; Protein Overexpression; Proteins; Proteolysis; Reporter; Research; Resistance profile; Solid; Solubility; Specificity; Structure-Activity Relationship; System; Technology; Testing; Therapeutic; Tissues; Tracer; Ubiquitination; assay development; base; cancer testis antigen; cancer therapy; carboxyfluorescein; cytotoxicity; dimer; experimental study; fluorophore; improved; in vivo; innovation; interest; knock-down; loss of function; male; melanoma; member; mutant; neoplastic cell; novel therapeutics; overexpression; peptidomimetics; protein degradation; recruit; small molecule; tumor; tumor specificity; tumorigenesis; ubiquitin-protein ligase

Project Summary: Proteolysis Targeting Chimeras (PROTACs) have introduced a new pharmacological paradigm, event-driven pharmacology. PROTACs are bifunctional small-molecules that simultaneously engage an E3 ubiquitin ligase and a protein of interest (POI). Ternary formation induced by PROTAC binding results in ubiquitination of the POI by the E3 ligase and subsequent degradation of the POI by the 26S proteasome. Most research efforts have focused on increasing POI diversity. However, identification and development of E3 ligase recruiting elements (E3REs) has lagged. The next innovation for PROTAC technology is the induction of tumor-specific protein degradation. PROTACs that induce degradation only in tumor cells are likely to have decreased off-target cytotoxicity, thereby improving their therapeutic utility. However, the E3 ligases most commonly recruited, Von Hippel-lindau, Cereblon, and Mouse double minute 2 homolog, are expressed in both cancerous and untransformed tissues. New E3REs must be developed that engage E3 ligases with tumor specific expression to impart tumor-specificity. Type I Melanoma Antigen Gene (MAGE) family proteins are cancer testis antigens, whose expression is restricted to the male germ line, but can be re-expressed in cancers. MAGE-A3 binds TRIM28, a ubiquitously expressed protein with E3 ligase activity, to form an oncogenic tumor-specific E3 ligase complex. A PROTAC harboring a MAGE-A3 E3RE may be able to recruit MAGE-A3/TRIM28 and induce tumor-specific degradation. MAGE-A3 has been found to exist as a dimer in solution. A fluorescent peptide that mimics key residues involved in this dimerization will be used to develop a fluorescence polarization assay (FP). The FP assay will then be used to identify a small-molecule ligand of MAGE-A3. Orthogonal biophysical assays, like thermal shift, intrinsic Trp fluorescence, and isothermal calorimetry will then be used to confirm binding. Once identified, the MAGE-A3 ligand will be used as an E3RE in the synthesis of a MAGE-A3 based PROTAC. Cellular experiments using the HaloTag7-GFP reporter system developed in the Crews Lab will then be used to test the activity of MAGE-A3 based PROTACs. This project will determine if a MAGE-A3 ERE3 can be used to recruit the MAGE-A3/TRIM28 E3 ligase complex to induce tumor-specific protein degradation. PROTACs created during this project may serve as the starting point for the future development of a tumor-specific therapy.

项目摘要： 靶向嵌合体的蛋白水解（Protac）引入了新的药理范式，事件驱动 药理。 protac是双功能的小分子，同时参与E3泛素连接酶 和感兴趣的蛋白质（POI）。由Protac结合诱导的三元形成导致泛素化 E3连接酶的POI以及随后由26S蛋白酶体对POI的降解。大多数研究工作 专注于增加POI多样性。但是，识别和开发E3连接酶募集元件 （e3res）滞后。 Protac技术的下一个创新是诱导肿瘤特异性蛋白 降解。仅在肿瘤细胞中诱导降解的protac可能会降低脱靶降低 细胞毒性，从而改善其治疗效用。但是，E3连接酶最常见的是von Hippel-Lindau，Cereblon和Mouse Double 2 Minuse 2同源物在癌症和 未转化的组织。必须开发新的E3RES，使E3连接酶具有特定于肿瘤的表达 赋予肿瘤特异性。 I型黑色素瘤抗原基因（MAGE）家族蛋白是癌症抗原，其表达是 仅限于男性生殖系，但可以在癌症中重新表达。 Mage-A3绑定Trim28，一种普遍存在的 表达具有E3连接酶活性的蛋白质，形成了致癌性肿瘤特异性E3连接酶复合物。 protac 拥有法师A3 E3RE可能能够招募法师A3/TRIM28并诱导肿瘤特异性降解。 MAGE-A3已被发现作为溶液中的二聚体存在。荧光肽模仿涉及的关键残基 在此二聚体中，将用于开发荧光极化测定（FP）。 FP测定将是 用于识别法师A3的小分子配体。正交生物物理测定，例如热移，内在 然后将使用TRP荧光和等温量热法来确认结合。 一旦确定，MAGE-A3配体将在基于MAGE-A3的Protac的合成中用作E3RE。 然后，使用在机组人员实验室中开发的halotag7-GFP报告基因系统的蜂窝实验将用于 测试基于MAGE-A3的Protac的活性。该项目将确定是否可以使用Mage-A3 ERE3 募集MAGE-A3/TRIM28 E3连接酶复合物以诱导肿瘤特异性蛋白质降解。 protacs 在该项目期间创建的可能是肿瘤特异性疗法未来发展的起点。