Targeted degradation of RNAs by using small molecules

使用小分子靶向降解 RNA

基本信息

批准号：
10661487
负责人：
Matthew D Disney
金额：
$ 66.16万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10661487
关键词：
Animals Antisense Oligonucleotides Apoptosis Base Sequence Benchmarking Binding Binding Proteins Biogenesis Breast Cancer Cell Breast Cancer Model Breast Cancer Patient Breast Cancer cell line Breast Epithelial Cells Cancer Center Cell Proliferation Cells Chimera organism Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complementary RNA Development Disease Engineering Evaluation Genetic Transcription Goals Human In Situ In Vitro Invaded Laboratories Lead MDA MB 231 Malignant Neoplasms Medicine Methods MicroRNAs Modality Mus Mutation Neoplasm Metastasis Normal tissue morphology Oligonucleotides Oncogenic Patients Pharmaceutical Preparations Phenotype Production Proliferating Property Protac Protein Inhibition Proteins Proteome RNA RNA Binding RNA Degradation RNA Splicing Resources Ribonuclease H Ribonucleases Role Safety Site Testing Triage Tumor Suppressor Proteins Tumor Tissue Untranslated RNA Validation Xenograft Model derepression design efficacy evaluation efficacy testing improved in vivo in vivo evaluation knock-down lead optimization malignant breast neoplasm migration neoplastic cell novel therapeutic intervention nuclease programs rational design recruit relapse prevention response small molecule stem cell biomarkers targeted treatment tool transcriptome triple-negative invasive breast carcinoma tumor tumorigenesis

项目摘要

We propose a transformative approach, inspired by the mechanism of action of antisense oligonucleotides (ASOs), to deliver small molecules that selectively cleave RNA targets in cells and in animals. As a state-of-the- art modality to target RNA, ASOs bind to complementary RNAs and recruit endogenous RNase H, which then cleaves the RNA to eliminate it from the cell. As an alternative to ASOs, we have developed a class of small molecules that selectively bind to and cleave an RNA target and have shown that our new cleaving small molecules are more potent than simple binding compounds. Our approach, dubbed Ribonuclease targeting chimeras (RIBOTACs), engineers small molecules to recruit endogenous RNase L, an RNase expressed at minute levels in cells in a latent form (hence RNase L). The chimeras, comprised of RNA-binding modules and a heterocyclic RNase L-recruiting module, activate RNase L locally at the site of the desired target. We will fully develop our RIBOTAC approach to cleave RNA targets sub-stoichiometrically and catalytically with small molecules, providing a direct means to improve the potency of simple binding compounds. Collectively, we will deliver a platform to program small molecules to cleave specific, malfunctioning RNAs in cells and in animals, with superior properties as compared to ASOs. In support of these goals, we propose in Aim 1 to characterize comprehensively our lead RIBOTAC targeting miR-21 in vitro and in situ, a benchmark for lead optimization. Our new studies show that the binding compound from which this RIBOTAC is derived inhibits metastasis in an orthotopic xenograft model. Further, the RIBOTAC is 20-fold more potent than the simple binding compound in situ for inhibiting miR-21 biogenesis and breast cancer cell phenotypes. Of import, we will study and quantify the selectivity of the RIBOTAC transcriptome- and proteome-wide. In Aim 2, we will lead optimize our RIBOTAC to deliver a proof-of-concept compound with properties amenable for in vivo testing. These DMPK-driven studies will optimize all components of the RIBOTAC, from the RNA-binding modules to the linker that tethers them together to the RNase L-recruiting module. We will rigorously assess top RIBOTACs in the triple negative breast cancer (TNBC) cell line MDA- MB-231, including full assessment and quantification of selectivity transcriptome- and proteome-wide. Finally, in Aim 3, we will study optimized RIBOTACs for activity against a panel of TNBC and patient-derived (PDX) tumor cells ex vivo and in vivo. After confirming miR-21 destruction by our RIBOTACs, we will assess their effects on TNBC cell: (i) proliferation; (ii) survival; (iii) migration and invasion; and (iv) expression of EMT and breast cancer stem cell markers. RIBOTACs with the broadest activity against TNBCs will be evaluated for efficacy in vivo.

受反义寡核苷酸作用机制的启发，我们提出了一种变革性方法 (ASO)，传递小分子，选择性切割细胞和动物中的 RNA 靶点。作为一个国家的 ASOs 与互补 RNA 结合并招募内源性 RNase H，然后切割 RNA，将其从细胞中消除。作为 ASO 的替代方案，我们开发了一类小型选择性结合并切割 RNA 靶标的分子，并表明我们的新切割小分子分子比简单的结合化合物更有效。我们的方法被称为核糖核酸酶靶向嵌合体（RIBOTACs），设计小分子来招募内源性 RNase L，一种在以下位置表达的 RNase：细胞中以潜在形式存在的微小水平（因此称为 RNase L）。嵌合体由 RNA 结合模块和杂环 RNase L 募集模块，在所需目标位点局部激活 RNase L。我们将全力开发我们的 RIBOTAC 方法，以亚化学计量和催化方式切割 RNA 靶标分子，提供了提高简单结合化合物效力的直接方法。我们将共同提供一个平台来对小分子进行编程，以切割细胞和动物中特定的、有故障的 RNA，与 ASO 相比具有优越的性能。为了支持这些目标，我们在目标 1 中建议全面描述我们的领先 RIBOTAC 目标 miR-21 体外和原位，先导化合物优化的基准。我们的新研究表明，结合化合物这种 RIBOTAC 的来源可抑制原位异种移植模型中的转移。此外，RIBOTAC 抑制 miR-21 生物发生和乳腺的作用比原位简单结合化合物强 20 倍癌细胞表型。重要的是，我们将研究和量化 RIBOTAC 转录组的选择性蛋白质组范围内。在目标 2 中，我们将主导优化我们的 RIBOTAC，以提供具有以下特性的概念验证化合物：适合体内测试的特性。这些 DMPK 驱动的研究将优化该药物的所有成分 RIBOTAC，从 RNA 结合模块到将它们连接到 RNase L 招募的接头模块。我们将严格评估三阴性乳腺癌 (TNBC) 细胞系 MDA- 中的顶级 RIBOTAC MB-231，包括转录组和蛋白质组选择性的全面评估和定量。最后，在目标 3 中，我们将研究优化的 RIBOTACs 针对一组 TNBC 和患者源性 (PDX) 的活性离体和体内肿瘤细胞。在确认我们的 RIBOTAC 破坏 miR-21 后，我们将评估其对TNBC细胞的影响：（i）增殖； (二) 生存； (iii) 迁移和入侵； (iv) EMT 的表达和乳腺癌干细胞标志物。将评估针对 TNBC 具有最广泛活性的 RIBOTAC 体内功效。