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

• 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
• 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' 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

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- 寡核苷酸结合物 摘要 我们建议合并小分子和反义寡核苷酸(ASO)的方法来特异性地抑制病毒 RNA翻译而不抑制内源性细胞mRNAs的翻译。罗加拉特天然产品， 如罗格列胺A(ROC)，展示了一种独特的RNA靶向机制，通过这种机制，小分子结合 到eIF4A解旋酶和多嘌呤RNA(A和G的延伸部分)之间的双分子空腔产生立体 夹住靶mRNAs的5‘非翻译区(UTRs)。由于扫描核糖体受阻，这 洛格列胺介导的钳制导致对含有多尿路的mRNAs的翻译抑制，包括 SARS-CoV-2和其他阳性RNA病毒的感染。而临床前的病毒复制分析表明 佐替芬是一种基于洛格拉胺的临床候选药物，它可能显示出一个很有希望的治疗窗口 作为一种抗病毒药物，预计同时抑制内含多聚尿路的mRNAs 这种细胞可能会产生剂量限制的毒性。 我们将洛格列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 通过附加的RNA靶向化学元素进行序列测定。我们设计了 洛格拉米特-ASO(RocASO)分子，将洛格拉胺与ASO连接，ASO的结合依赖于 与靶RNA序列的互补碱基配对。将两种治疗方式并列的模型 显示了两个绑定元素的允许配置，它们相隔大约20？，它们将被遍历 通过足够长度的可变化学系绳。针对SARS-CoV-2的5‘非编码区设计的RocASO将是 使用eIF4A和病毒RNA的生物化学三元复合体形成试验以及使用 SARS-CoV-2 5‘端非编码区翻译与RNA稳定性的细胞报道最后，我们将在一个 用SARS-CoV-2复制子系统测定ROASO对多尿路或ASO的相对耐受性 识别序列突变。这些实验还将评估潜在的新变化 靶向序列可以通过对RocASO引入伴随的修饰、评估 平台作为新型SARS-CoV-2变异体和其他病毒5‘非编码区的适应性RNA靶向方式 序列。