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.

项目概要： 蛋白水解靶向嵌合体（PROTAC）引入了一种新的药理学范式，事件驱动 药理学PROTAC是同时与E3泛素连接酶结合的双功能小分子 和感兴趣的蛋白质（POI）。由PROTAC结合诱导的三元体形成导致蛋白质的泛素化。 通过E3连接酶降解POI，随后通过26 S蛋白酶体降解POI。大多数研究工作 专注于增加POI的多样性。然而，E3连接酶募集元件的鉴定和开发， （E3 RE）已经落后了。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可能具有减少的脱靶 细胞毒性，从而提高其治疗效用。然而，最常募集的E3连接酶，Von Hippel-lindau、Cereblon和小鼠双微体2同源物在癌性和非癌性细胞中表达。 未转化的组织必须开发新的E3 RE，使E3连接酶与肿瘤特异性表达结合 以赋予肿瘤特异性。 I型黑素瘤抗原基因（法师）家族蛋白是癌症睾丸抗原，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结合TRIM 28，一种普遍存在的 表达的具有E3连接酶活性的蛋白，以形成致癌肿瘤特异性E3连接酶复合物。一种PROTAC 携带MAGE-A3 E3 RE的细胞可以募集MAGE-A3/TRIM 28并诱导肿瘤特异性降解。 已发现MAGE-A3在溶液中作为二聚体存在。一种模拟关键残基的荧光肽， 在这种二聚化中，将用于开发荧光偏振测定（FP）。然后将进行FP测定， 用于鉴定MAGE-A3的小分子配体。正交生物物理分析，如热位移，内在 然后使用Trp荧光和等温量热法确认结合。 一旦鉴定，MAGE-A3配体将在基于MAGE-A3的PROTAC的合成中用作E3 RE。 然后将使用在Crews实验室开发的HaloTag 7-GFP报告系统进行细胞实验， 测试基于MAGE-A3的PROTAC的活性。本项目将确定MAGE-A3 ERE 3是否可用于 募集MAGE-A3/TRIM 28 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新的肿瘤特异性疗法可以作为未来肿瘤特异性疗法开发的起点。