Targeting Viral RNA Using a Sequence Programmable Small Molecule-Oligonucleotide Conjugate

使用序列可编程小分子-寡核苷酸缀合物靶向病毒 RNA

基本信息

批准号：
10512627
负责人：
KEVAN M. SHOKAT
金额：
$ 545.04万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-16 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10512627
关键词：
2019-nCoV 5&apos Untranslated Regions Address Affinity Antisense Oligonucleotides Base Pairing Binding Biochemical Biological Assay COVID-19 treatment Cells Chemicals Chikungunya virus Clinic Closure by clamp Collaborations Communicable Diseases Complex Coronavirus Crimean-Congo Hemorrhagic Fever Virus Cryoelectron Microscopy Crystallization Dose Dose-Limiting Elements Engineering Glues Head Human poliovirus In Vitro Laboratories Lassa virus Lead Length Letters Ligands Link Linker-Oligonucleotide Measures Mediating Messenger RNA Middle East Respiratory Syndrome Coronavirus Modality Modeling Modification Molecular Monitor Mutation Natural Products Obstruction Oligonucleotides Pharmaceutical Preparations Positioning Attribute Proteins Proteomics RNA RNA Sequences RNA Stability RNA Viruses Replicon Reporter Research Personnel Ribosomes Role SARS coronavirus SARS-CoV-2 variant Scanning Series Site Specificity Stretching Structure System Testing Therapeutic Toxic effect Traction Translational Repression Translations Variant Viral Viral Genome Viral Proteins Virus Virus Replication Zika Virus appendage arm base chemical genetics clinical candidate design experimental study helicase in vitro Assay in vivo inhibitor novel pandemic disease pre-clinical programs response rocaglamide small molecule structural biology translation factor viral RNA virology

项目摘要

PROJECT 1: TARGETING VIRAL RNA USING A SEQUENCE PROGRAMMABLE SMALL MOLECULE- OLIGONUCLEOTIDE CONJUGATE SUMMARY We propose to merge small molecule and antisense oligonucleotide (ASO) approaches to specifically inhibit viral RNA translation without inhibiting the translation of endogenous cellular mRNAs. Rocaglate natural products, such as rocaglamide A (Roc), demonstrate a unique RNA-targeting mechanism by which small molecule binding to a bimolecular cavity between eIF4A helicase and polypurine RNA (stretches of A and G) generates a steric clamp on the 5’ untranslated regions (UTRs) of target mRNAs. Due to obstruction of the scanning ribosome, this rocaglamide-mediated clamp results in translational inhibition of polypurine tract-containing mRNAs, including that of SARS-CoV-2 and other positive-sense RNA viruses. While pre-clinical viral replication assays indicate that zotatifin, a clinical candidate based on rocaglamide, may demonstrate a promising therapeutic window as an anti-viral, it is anticipated that the simultaneous inhibition of multiple polypurine tract-containing mRNAs within the cell could result in dose-limiting toxicities. We position rocaglamide as a unique protein-RNA molecular glue that can be directed to specific viral target sequences through the appendage of an additional RNA-targeting chemical element. We have designed rocaglamide-ASO (RocASO) molecules which link rocaglamide to an ASO whose binding is dependent upon complementary base-pairing with target RNA sequences. A model that juxtaposes the two therapeutic modalities reveals a permissible disposition of the two binding elements approximately 20 Å apart, which will be traversed by a variable chemical tether of sufficient length. RocASOs designed against the 5’ UTR of SARS-CoV-2 will be tested using biochemical ternary complex formation assays with eIF4A and viral RNA, as well as assayed using cellular reporters of SARS-CoV-2 5’ UTR translation and RNA stability. Finally, we will apply RocASOs in a SARS-CoV-2 replicon system to determine the relative tolerance of RocASOs to polypurine tract or ASO recognition sequence mutations. These experiments will also evaluate whether potentially emergent alterations to target sequences could be addressed by introducing concomitant modifications to RocASOs, assessing the platform as an adaptable RNA targeting modality for novel SARS-CoV-2 variants and other viral 5’ UTR sequences.

项目 1：使用序列可编程小分子靶向病毒 RNA - 寡核苷酸缀合物概括我们建议合并小分子和反义寡核苷酸（ASO）方法来特异性抑制病毒 RNA 翻译，不抑制内源细胞 mRNA 的翻译，Rocaglate 天然产物。例如 rocaglamide A (Roc)，展示了一种独特的 RNA 靶向机制，通过该机制小分子结合 eIF4A 解旋酶和多聚嘌呤 RNA（A 和 G 的延伸）之间的双分子腔产生空间立体由于扫描核糖体受到阻碍，因此会夹住目标 mRNA 的 5' 非翻译区 (UTR)。罗卡拉酰胺介导的钳夹导致含有多嘌呤束的 mRNA 的翻译抑制，包括 SARS-CoV-2 和其他正义 RNA 病毒的临床前病毒复制测定表明。佐他替芬（zotatifin）是一种基于罗卡酰胺的临床候选药物，可能会显示出一个有希望的治疗窗口：一种抗病毒药物，预计同时抑制体内多个含有多聚嘌呤束的 mRNA 细胞可能会导致剂量限制性毒性。我们将罗卡酰胺定位为一种独特的蛋白质-RNA分子胶，可以针对特定的病毒靶点我们设计了一个附加的 RNA 靶向化学元素的序列。罗卡酰胺-ASO (RocASO) 分子，将罗卡酰胺与 ASO 连接，其结合取决于与靶 RNA 序列的互补碱基配对并列两种治疗方式的模型。揭示了两个相距约 20 Å 的结合元素的允许配置，这将被遍历由针对 SARS-CoV-2 5'UTR 设计的足够长度的可变化学链将是使用 eIF4A 和病毒 RNA 的生化三元复合物形成测定进行测试，并使用 SARS-CoV-2 5' UTR 翻译和 RNA 稳定性的细胞生产最后，我们将在 a 中应用 RocASO。 SARS-CoV-2 复制子系统，用于确定 RocASO 对多聚嘌呤束或 ASO 的相对耐受性这些实验还将评估是否存在潜在的紧急改变。可以通过对 RocASO 进行伴随修改来解决对目标序列的影响，评估平台作为新型 SARS-CoV-2 变体和其他病毒 5' UTR 的适应性 RNA 靶向方式序列。