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：使用可编程的小分子靶向病毒RNA 寡核苷酸结合概括我们建议将小分子和反义寡核苷酸（ASO）合并以特异性抑制病毒 RNA翻译而不抑制内源性细胞mRNA的翻译。 Rocaglade天然产品，例如Rocaglamide A（ROC），展示了一种独特的RNA靶向机制，小分子结合 EIF4A解旋酶和息肉RNA（a和g的拉伸）之间的双分子腔产生一个空间在目标mRNA的5'未翻译区域（UTRS）上夹紧。由于扫描核糖体的阻塞，这 Rocaglamide介导的夹具会导致转化息肉素段含有息肉的mRNA，包括 SARS-COV-2和其他阳性RNA病毒。而临床前病毒复制分析表明 Zotatifin是基于Rocaglamide的临床候选者，可能表现出有希望的治疗窗口抗病毒，预计简单地抑制了多种息肉素含有含有息肉的mRNA 该细胞可能导致剂量限制性毒性。我们将Rocaglamide定位为一种独特的蛋白RNA分子胶，可以针对特定病毒靶标序列通过附加靶向RNA的化学元件的序列。我们已经设计了 Rocaglamide-Aso（Rocaso）分子，将Rocaglamide与ASO联系起来，其结合取决于与目标RNA序列完全基本对。将两种治疗方式并列的模型揭示了大约20Å的两个结合元件的许可处置通过足够长度的可变化学系列。针对SARS-COV-2的5'UTR设计的Rocasos将是使用EIF4A和病毒RNA的生化三元化合物形成测定测试，并使用使用 SARS-COV-2 5'UTR翻译和RNA稳定性的细胞记者。最后，我们将在一个 SARS-COV-2复制品系统，以确定Rocasos对息肉素或ASO的相对耐受性识别序列突变。这些实验还将评估是否存在潜在的紧急改变可以通过对Rocasos引入伴随的修改来解决目标序列，并评估平台作为新型SARS-COV-2变体和其他病毒5'UTR的适应性RNA靶向方式序列。