Targeted degradation of RNAs by using small molecules

我们提出了一种变革性的方法，受到反义寡核苷酸作用机制的启发， （AS 0），以递送选择性地切割细胞和动物中的RNA靶标的小分子。作为一个国家的- 作为靶向RNA的一种方式，ASO与互补RNA结合并募集内源性RNA酶H，然后 切割RNA将其从细胞中清除。作为ASO的替代品，我们开发了一类小型 选择性结合并切割RNA靶分子，并已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），工程师小分子招募内源性RNase L，一种表达于 在潜伏形式的细胞中的微小水平（因此RNase L）。嵌合体由RNA结合模块组成， 杂环RNA酶L-募集模块，在所需靶位点局部激活RNA酶L。全面 开发我们的RIBOTAC方法，以亚化学计量和催化方式切割RNA靶点， 分子，提供了一种直接的手段，以提高简单的结合化合物的效力。我们将共同 提供一个平台来编程小分子，以切割细胞和动物中特定的故障RNA， 与ASO相比具有上级性能。 为了支持这些目标，我们在目标1中建议全面描述我们的主要RIBOTAC靶向 miR-21在体外和原位，铅优化的基准。我们的新研究表明 该RIBOTAC的来源抑制原位异种移植模型中的转移。此外，RIBOTAC 在抑制miR-21生物发生和乳腺癌方面， 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。在目标2中，我们将率先优化我们的RIBOTAC，以提供一种概念验证化合物， 适合于体内测试的性质。这些DMPK驱动的研究将优化 RIBOTAC，从RNA结合模块到将它们拴在一起的接头，再到RNA酶L-募集 module.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，包括选择性转录组和蛋白质组范围的全面评估和定量。最后， 在目标3中，我们将研究优化的RIBOTAC对一组TNBC和患者来源的（PDX）的活性， 离体和体内的肿瘤细胞。在确认我们的RIBOTAC破坏miR-21后，我们将评估它们的 对TNBC细胞的影响：（i）增殖;（ii）存活;（iii）迁移和侵袭;和（iv）EMT和EMT的表达。 乳腺癌干细胞标志物将评价对TNBC具有最广泛活性的RIBOTAC， 体内功效

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.