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.

我们提出了一种转化方法，灵感来自反义寡核苷酸的作用机理（ASO），输送小分子，在细胞和动物中有选择地切割RNA靶标。作为最先进的靶向RNA的ART模态，ASO与互补的RNA结合并募集内源性RNase H，然后切割RNA以将其从细胞中消除。作为ASOS的替代方案，我们已经开发了一类小型选择性结合并裂解RNA靶的分子，并表明我们的新裂解小分子比简单结合化合物更有效。我们的方法称为核糖核酸酶的靶向嵌合体（Ribotacs），工程师小分子以募集内源性RNase L，一种在细胞中的微小水平（因此是RNase L）。嵌合体由RNA结合模块和杂环RNase l恢复模块在所需目标的位置局部激活RNase L。我们将完全开发我们的Ribotac方法，以小型计量和催化的方式裂解RNA靶标。分子提供了一种直接的手段来提高简单结合化合物的效力。总体而言，我们会的提供一个平台以编程小分子以裂解特定的细胞和动物中的RNA，在动物中，与ASO相比，具有出色的性质。为了支持这些目标，我们在AIM 1中提出，以全面表征我们的Ribotac目标 miR-21在体外和原位，这是铅优化的基准。我们的新研究表明结合化合物从中得出该肋骨可在原位异种移植模型中抑制转移。此外，Ribotac 比简单结合化合物原位抑制miR-21生物发生和乳房的效力要高20倍癌细胞表型。进口，我们将研究和量化Ribotac转录组和的选择性全蛋白质组。在AIM 2中，我们将领导优化我们的Ribotac，以提供概念验证的化合物可用于体内测试的特性。这些DMPK驱动的研究将优化 Ribotac，从RNA结合模块到接头，将它们绑定到RNase l-Recruiting 模块。我们将严格评估三重阴性乳腺癌（TNBC）细胞系MDA- MB-231，包括对整个蛋白质组的选择性转录组和全蛋白质组的全面评估和定量。最后，在AIM 3中，我们将研究针对TNBC和患者衍生（PDX）活动的优化Ribotacs。肿瘤细胞离体和体内。在确认了Ribotacs的MiR-21破坏之后，我们将评估他们的对TNBC细胞的影响：（i）增殖；（ii）生存；（iii）迁移和入侵；（iv）EMT和乳腺癌干细胞标记。将评估针对TNBC的最广泛活动的Ribotacs 体内功效。