Developing Tumor-specific PROTACs

开发肿瘤特异性 PROTAC

• 批准号: 10244943
• 负责人: CRAIG M CREWS
• 金额: $ 38.22万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-05 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10244943
• 关键词: 26S proteasome; 5' -AMP-activated protein kinase; Affinity; Antigens; Antineoplastic Agents; Apoptosis; Binding; Binding Proteins; Biological Assay; Biophysics; Bromodomain; Calorimetry; Cancer Patient; Cancerous; Cell Culture Techniques; Cell Death; Cells; Complex; Computer software; Crystallization; Dependence; Development; Docking; Dose; Double Minutes; Drug resistance; Elements; Event; Exhibits; Flow Cytometry; Fluorescein; Fluorescence; Fluorescence Polarization; Future; Gene Expression; Gene Family; Gene Proteins; Genes; Germ Lines; Homologous Gene; Immunoblotting; Induction of Apoptosis; Lead; Libraries; Ligands; Malignant Neoplasms; Measures; Mediating; Mus; Oncogenic; Protac; Protein Family; Proteins; Reporter; Research; Resistance profile; Solubility; Structure; Structure-Activity Relationship; System; Technology; Testing; Therapeutic; Tissues; Tryptophan; Ubiquitination; base; biophysical techniques; c-myc Genes; cancer cell; cancer testis antigen; cytotoxicity; design; functional group; improved; in silico; in vivo; innovation; insight; interest; male; melanoma; member; mutant; neoplastic cell; novel; programs; protein degradation; recruit; small molecule; tumor; tumor specificity; tumorigenesis; ubiquitin-protein ligase; vector

Proteolysis Targeting Chimeras (PROTACs) are event-driven bifunctional small-molecules that simultaneously engage an E3 ubiquitin ligase and a protein of interest (POI). Ternary complex formation between POI-PROTAC- E3 ligase, results in E3-ligase mediated POI ubiquitination and subsequent degradation of the POI by the 26S proteasome. The next innovation for PROTAC technology is the induction of tumor-specific protein degradation. PROTACs that induce degradation selectively in tumor cells would likely have improved therapeutic utility due to decreased off-target cytotoxicity. Currently, the E3 ligases most commonly hijacked for PROTAC-mediated POI degradation, von Hippel-Lindau, Cereblon, and Mouse double minute 2 homolog, are expressed in both cancerous and untransformed tissues. Therefore, new E3 ligase recruiting elements (E3REs) that engage E3 ligases with tumor-specific expression must be developed 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 protein degradation in a tumor-specific manner. MAGE proteins bind their cognate E3 ligases and substrates via a conserved MAGE homology domain (MHD). Using Schrödinger Glide docking software, we screened >60,000 compounds against the recently resolved structure of the MAGE-A3 MHD to identify ligands in silico that are predicted to disrupt MAGE-A3- substrate binding. We have identified a subset of lead-like compounds using intrinsic tryptophan fluorescence and are currently corroborating these findings via orthogonal biophysical assays such as isothermal calorimetry, and various NMR-based strategies. A structure-activity relationship study on bona-fide MAGE-A3 binders will then be performed to improve solubility, increase affinity, and identify (a) potential vector(s) for linker attachment in subsequent PROTAC development. Once tight-binding MAGE-A3 ligands have been developed, we will synthesize MAGE-A3-based-HaloPROTACs and test their ability to degrade HaloTag7-GFP in a MAGE-A3- dependent manner. Subsequently, we will further test the utility of recruiting MAGE-A3/TRIM28 E3 ligase complex by targeting Bromodomain-containing protein 4 (BRD4) for MAGE based-PROTAC mediated degradation. Induction of tumor-specific degradation of BRD4 and induction of apoptosis in a tumor-specific manner by our MAGE-A3 based-PROTACs will be evaluated. Overall, this project will determine the MAGE- A3/TRIM28 E3 ligase complex induce tumor-specific protein degradation. Additionally, development of a new E3RE will help spark excitement for identification of novel E3REs for other E3 ligases, thereby greatly expanding the number of E3 ligase amenable to the PROTAC technology. Moreover, PROTACs created during this project may serve as the starting point for the future development of a tumor-specific therapy.

蛋白水解靶向嵌合体 (PROTAC) 是事件驱动的双功能小分子，同时 连接 E3 泛素连接酶和目标蛋白 (POI)。 POI-PROTAC-之间的三元复合物形成 E3 连接酶，导致 E3 连接酶介导的 POI 泛素化以及随后 26S 对 POI 的降解 蛋白酶体。 PROTAC 技术的下一个创新是诱导肿瘤特异性蛋白 降解。在肿瘤细胞中选择性诱导降解的 PROTAC 可能会得到改善 由于脱靶细胞毒性降低而具有治疗效用。目前，E3 连接酶最常被劫持 PROTAC 介导的 POI 降解、von Hippel-Lindau、Cereblon 和小鼠双分钟 2 同源物是 在癌组织和未转化组织中表达。因此，新的 E3 连接酶招募元件 (E3RE) 必须开发与肿瘤特异性表达结合的 E3 连接酶，以赋予肿瘤特异性。 Ⅰ型 黑色素瘤抗原基因 (MAGE) 家族蛋白是癌症睾丸抗原，其表达仅限于 男性生殖系，但可以在癌症中重新表达。 MAGE-A3 结合 TRIM28，TRIM28 是一种普遍表达的 具有E3连接酶活性的蛋白质，形成致癌肿瘤特异性E3连接酶复合物。一个 PROTAC 包含一个 MAGE-A3 E3RE 可能能够招募 MAGE-A3/TRIM28 并诱导肿瘤特异性蛋白降解 方式。 MAGE 蛋白通过保守的 MAGE 同源结构域结合其同源 E3 连接酶和底物 （磁流体动力学）。使用 Schrödinger Glide 对接软件，我们针对最近的化合物筛选了超过 60,000 种化合物 解析 MAGE-A3 MHD 的结构，以在计算机中识别预计会破坏 MAGE-A3 的配体- 底物结合。我们利用内在色氨酸荧光鉴定了类铅化合物的子集 目前正在通过等温量热法等正交生物物理测定法证实这些发现， 以及各种基于 NMR 的策略。对真正的 MAGE-A3 结合物的结构-活性关系研究将 然后进行以提高溶解度、增加亲和力并鉴定用于接头连接的潜在载体 在随后的PROTAC开发中。一旦开发出紧密结合的 MAGE-A3 配体，我们将 合成基于 MAGE-A3 的 HaloPROTAC 并测试其在 MAGE-A3 中降解 HaloTag7-GFP 的能力 依赖方式。随后，我们将进一步测试招募MAGE-A3/TRIM28 E3连接酶的效用 通过针对基于 MAGE 的 PROTAC 介导的含溴结构域蛋白 4 (BRD4) 的复合物 降解。诱导肿瘤特异性 BRD4 降解并诱导肿瘤特异性细胞凋亡 将通过我们基于 MAGE-A3 的 PROTAC 的方式进行评估。总的来说，这个项目将决定 MAGE- A3/TRIM28 E3 连接酶复合物诱导肿瘤特异性蛋白质降解。此外，还开发了一种新的 E3RE 将有助于激发对其他 E3 连接酶的新型 E3RE 的鉴定，从而极大地扩展 适合 PROTAC 技术的 E3 连接酶的数量。此外，在该项目期间创建的 PROTAC 可能作为未来开发肿瘤特异性疗法的起点。