Developing Tumor-specific PROTACs

开发肿瘤特异性 PROTAC

基本信息

批准号：
10470405
负责人：
CRAIG M CREWS
金额：
$ 37.44万
依托单位：
YALE UNIVERSITY
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：
2019-09-05 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10470405
关键词：
26S proteasome 5&apos -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 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 Western Blotting 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

26S proteasome 5&apos -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 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 Western Blotting 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

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项目摘要

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.

靶向嵌合体的蛋白水解是事件驱动的双功能小分子，简单参与E3泛素连接酶和感兴趣的蛋白质（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泛素化和随后的POI降解26S 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降解的protac可能会改善由于脱靶细胞毒性降低而导致的治疗效用。目前，E3连接酶最常被劫持 Protac介导的POI降解，von Hippel-Lindau，Cereblon和Mouse Double 2同源物在取消和未转化的组织中表达。因此，新的E3连接酶招募元素（E3RES）必须开发具有肿瘤特异性表达的E3连接酶以赋予肿瘤特异性。类型I。黑色素瘤抗原基因（MAGE）家族蛋白是癌睾丸抗原，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Trim28，一种普遍表达的具有E3连接酶活性的蛋白质，形成致癌性肿瘤特异性E3连接酶复合物。藏有一个的protac MAGE-A3 E3RE可能能够募集Mage-A3/TRIM28并在肿瘤特异性中衍生的蛋白质降解方式。法师蛋白通过配置的法师同源域结合其同源E3连接酶和底物（MHD）。使用SchrödingerGlide对接软件，我们针对最近的化合物进行了筛选> 60,000种化合物 MAGE-A3 MHD的解决结构，以识别有机硅中的配体，这些配体被预测会破坏Mage-A3- 底物结合。我们已经使用固有的色氨酸荧光确定了铅状化合物的子集目前正在通过正交生物物理测定（例如等温量热法）来证实这些发现，以及各种基于NMR的策略。对真正的法师A3粘合剂的结构活性关系研究将然后执行以提高溶解度，提高亲和力并确定（a）接头附件的潜在向量在随后的Protac开发中。一旦开发了紧密的法师A3配体，我们将合成基于法MAGE-A3的半甲酸，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法师A3/TRIM28 E3连接酶的实用性通过靶向含溴脱域的蛋白质4（BRD4）的复合物，用于法师基于PROTAC介导降解。诱导BRD4肿瘤特异性降解和肿瘤特异性诱导的凋亡将评估我们的MAGE-A3基于PROTAC的方式。总体而言，该项目将决定法师 - A3/TRIM28 E3连接酶复合物诱导肿瘤特异性蛋白质降解。此外，开发新的 E3RE将有助于引发兴奋，以识别其他E3连接酶的新型E3RES，从而大大扩展 Protac技术的E3连接酶的数量。而且，在这个项目中创建的Protac 可以作为肿瘤特异性疗法未来发展的起点。