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

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 总结 我们建议合并小分子和反义寡核苷酸（阿索）的方法，以特异性抑制病毒 RNA翻译而不抑制内源性细胞mRNA的翻译。Rocaglate天然产品， 例如罗格列酰胺A（Roc），证明了一种独特RNA靶向机制，通过该机制，小分子结合 eIF 4A解旋酶和多嘌呤RNA（A和G的延伸）之间的双分子空腔产生空间位阻。 夹在靶mRNA的5'非翻译区（UTR）上。由于扫描核糖体的阻碍， 罗格列胺介导的钳夹导致含有多嘌呤片段的mRNA的翻译抑制，包括 SARS-CoV-2和其他正义RNA病毒。虽然临床前病毒复制检测表明 佐他替芬是一种基于罗格列胺的临床候选药物， 作为一种抗病毒药物，可以预期，同时抑制多个含有多嘌呤片段的mRNA， 该细胞可能导致剂量限制性毒性。 我们将罗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标 通过附加的RNA靶向化学元件的附加物来修饰序列。我们设计了 罗格列酰胺-ASO（RocASO）分子，其将罗格列酰胺连接至阿索，所述ASO的结合依赖于 与靶RNA序列互补碱基配对。一种将两种治疗方式并置的模式 显示了两个装订元件相距约20 mm的允许布置， 由足够长的可变化学系链所控制针对SARS-CoV-2的5' UTR设计的RocASO将被 使用eIF 4A和病毒RNA的生物化学三元复合物形成测定进行测试，以及使用 SARS-CoV-2 5' UTR翻译和RNA稳定性的细胞报告基因。最后，我们将在一个 SARS-CoV-2复制子系统测定RocASO对多嘌呤段或阿索的相对耐受性 识别序列突变。这些实验还将评估潜在的突变是否 可以通过对RocASO引入伴随的修饰来解决靶序列， 作为新型SARS-CoV-2变体和其他病毒5' UTR的适应性RNA靶向模式的平台 序列的

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.