EIF4E

引言 信使RNA (mRNA) 作为一类新兴药物，因其优异的可编程性和安全性被广泛应用于疫苗，肿瘤免疫疗法，基因治疗和蛋白质替代疗法等重要研究领域。传统mRNA药物往往具有较短的半衰期和有限的蛋白生产能力，这限制了mRNA药物在具有更高治疗阈值疗法（如蛋白替代疗法）中的有效性。王潇团队近年致力于发展下一代mRNA设计及优化方法，课题组过去曾通过结合寡聚核糖核酸化学合成和mRNA酶合成方法，可控地在线性mRNA 3′端引入化学和拓扑学修饰以提高其功能稳定性，而翻译效率则是mRNA药物有效性的另一维度。 翻译起始是mRNA指导蛋白合成的限速步骤，在这一过程中，真核翻译起始因子（eIFs）通过与mRNA的N7-甲基-鸟嘌呤 (m7G) 帽结构相互作用进一步招募细胞质中的核糖体等内源蛋白翻译机器，从而开启mRNA翻译。大量此前研究表明，mRNA帽以及5′非翻译区域的化学修饰对于mRNA翻译及稳定性具有重要调控作用并展现出了改善治疗性mRNA药物性质的巨大潜力。尽管如此，现有基于RNA聚合酶的mRNA合成技术由于受到RNA合成酶结构兼容性的限制，难以引入更为多样化的mRNA化学或拓扑结构的修饰。与此同时，具有外切酶抗性的环状RNA (circRNA) 作为更稳定的治疗性蛋白表达载体近年来也收到了广泛关注。然而，环状RNA依赖于由内部核糖体进入位点 (IRES) 驱动的蛋白翻译效率远低于经典mRNA帽依赖的翻译，导致环状RNA的蛋白表达能力较低。因此，通过环状RNA拓扑结构改造引入帽结构有望在保持环状RNA稳定性的同时提高其翻译效率。 2024年9月23日，美国麻省理工学院化学系/Broad研究所王潇课题组在Nature Biotechnology发表了题为Chemical and topological design of multicapped mRNA and capped circular mRNA to augment translation 的研究论文。研究人员通过基于连接的mRNA-寡聚核苷酸组装策略（RNA LEGO），利用化学酶法实现了汇聚式mRNA模块化组装，在更大的化学空间上探索了mRNA 5′和3′以及环状RNA化学修饰和拓扑结构改造，大幅提高了mRNA在细胞与小鼠体内的蛋白生产能力，为mRNA翻译起始机制及相关化学修饰设计提供了新见解。 图1: LEGO，一种位点特异性对mRNA/circRNA进行化学/拓扑修饰的策略（Credit: Nature Biotechnology） 为了实现对mRNA及环状RNA的化学修饰及拓扑结构改造，王潇团队提出基于多种连接酶的mRNA-寡聚核苷酸组装策略（RNA LEGO），利用化学酶法实现了汇聚式mRNA模块化组装。首先，通过化学加帽法及反向高效液相色谱，研究者利用RNA LEGO策略合成并筛选了mRNA帽以及5′非翻译区域的多种天然或非天然化学修饰，并通过组合多种单一有效的化学修饰成功将荧光素酶报告mRNA的蛋白表达提高了近10倍。同时，基于事实上eIF在结合5′帽后，翻译起始复合体的组装完全依赖于蛋白-蛋白相互作用，课题组认为在5′非翻译区域引入非天然链接结构可能能被翻译起始机制兼容；与此同时，受课题组先前多尾RNA工作的启发，课题组推论在5′端引入多个帽结构以增加mRNA-eIF的结合位点可能加速翻译起始复合体的形成。基于这些假设，研究者结合化学加帽反应和点击化学组装策略构建了含有双帽结构的枝状寡聚核苷酸，并通过T4连接酶引入mRNA的5′末端，形成多帽mRNA，并发现其可以提高mRNA的蛋白表达。通过对多帽mRNA拓扑结构的改造优化，研究者确定了最佳的主链及支链长度，从而实现mRNA蛋白表达水平的最大化。最终，结合mRNA 5′ 化学修饰、双帽拓扑结构改造以及该课题组前期发展的mRNA 3′ 化学修饰策略，研究者将荧光素酶报告mRNA的峰值蛋白表达水平提高了近20倍。 图2: 多帽mRNA的设计思路（Credit: Nature Biotechnology） 为了研究优化后帽及邻近的化学/拓扑修饰增强翻译的机制，作者首先应用连接有含帽的寡多聚核糖核酸磁珠及同位素标记定量蛋白沉降质谱对可能的作用蛋白进行了全局表征。作者观察到包括帽结合蛋白eIF4E1（13倍）及其他翻译相关蛋白如eIF4G1（42倍），eIF4G3（28倍），eIF4A（6倍）的显著富集。除此之外，化学修饰只增强了翻译相关帽结合蛋白eIF4E1的富集，而对其余翻译不相关的帽结合蛋白如NCBP1的富集却下降了33倍，显示化学修饰对翻译相关机制的选择性。通过电泳迁移率检测实验，作者在体外用提纯蛋白确认了优化后的化学修饰或多帽结构能增强eIF4E1的结合，并且更显著的是，化学修饰也提高了帽-eIF4E-eIF4G共聚物的总体稳定性。除了结合翻译起始蛋白，帽结构同时也具有保护mRNA从5′端被外切酶降解的作用，这一过程被脱帽蛋白hDcp2调控。在这一方面，作者发现除了对帽本身的修饰，对5′非翻译区的化学修饰显著增强了mRNA对脱帽酶的抗性，最优修饰的mRNA在体外脱帽酶6小时的作用下仅被脱帽30%，而天然的帽0/1结构在同样条件下15分钟内就被完全脱帽。最后，通过课题组自主发明的空间转录/翻译组技术STARmap和RIBOmap，作者发现双帽mRNA在细胞内显著增加了核糖体结合的mRNA比例，验证了化学修饰及双帽结构双帽结构通过提高mRNA与翻译起始因子eIF4E的相互作用提高翻译起始速率进而提高翻译效率。 图3: 含帽的环状RNA的设计思路（Credit: Nature Biotechnology） 接下来，利用RNA LEGO的汇聚式合成策略，作者通过结合T4 RNA连接酶和RtcB连接酶介导的连接反应构建了一种不依赖于内含子的环状RNA环化及位点特异性修饰方法，首次合成了带有枝状帽结构的环状RNA并命名为”QRNA”。结合对枝状mRNA帽结构的化学修饰，研究者证明了环状RNA利用帽依赖机制进行翻译的可行性，并在体外和体内实现了对环状RNA翻译效率的大幅提高。通过对多种具有不同拓扑结构RNA翻译的研究，研究者提出了“帽临近驱动的翻译”模型：当5′帽以及非翻译序列通过天然磷酸二酯键、非天然共价或非共价互补配对等方式与mRNA相互作用时，在mRNA蛋白编码区附近的帽结构便可以招募核糖体并以“插入(slot-in)”的模式着陆于帽结构下游或上游约20碱基的mRNA编码区内并启动蛋白翻译。这一机制的揭示标明了mRNA翻译对各种不同拓扑结构的兼容性，并为后续的mRNA药物结构设计以及可能的天然mRNA翻译调控过程提供了新的启示。 图4: 帽临近驱动翻译模型（Credit: Nature Biotechnology） 最后，研究者分别测试了该技术在mRNA疫苗和蛋白替代疗法中的治疗效果。结果表明， 相比于未修饰的mRNA，经优化的编码人促红细胞生成素(hEPO)的双帽mRNA-LNP复合物能够在小鼠体内产生总共近8倍的hEPO蛋白表达并显著提高小鼠血液中网织红细胞的比例，显示其用于蛋白质疗法的优越性。而在针对SARS-CoV-2的mRNA疫苗应用中，经优化的mRNA不仅能够在注射后七天内产生抗体免疫反应，而且在加强针免疫一周后产生4倍于未修饰mRNA组的抗体滴度。通过运用空间转录/翻译组学技术对小鼠淋巴的表征显示，优化后mRNA显著提高了抗原呈递细胞的激活程度；通过流式细胞术对小鼠脾细胞的表征，优化后mRNA疫苗显著增加了T细胞响应，这些结果表明了化学修饰和拓扑结构改造在提高mRNA治疗应用方面的潜力。 参考文献 https://www.nature.com/articles/s41587-024-02393-y 责编|探索君 排版|探索君 文章来源|“BioArt” End 往期精选 围观 一文读透细胞死亡(Cell Death) | 24年Cell重磅综述（长文收藏版） 热文 Cell | 是什么决定了细胞的大小？ 热文 Nature | 2024年值得关注的七项技术 热文 Nature | 自身免疫性疾病能被治愈吗？科学家们终于看到了希望 热文 CRISPR技术进化史 | 24年Cell综述

DURHAM, N.C.--( BUSINESS WIRE )-- Ribometrix , a biotechnology company developing small molecule therapeutics that modulate RNA biology, presented the latest data from its eIF4E program at the European Society for Medical Oncology (ESMO) Congress, taking place in Barcelona, Spain, September 13-17. The poster presentation reviews in vitro and in vivo studies of a small molecule eIF4E inhibitor, RBX-6610, as a potential treatment for KRAS G12C mutant non-small cell lung cancer (NSCLC). Acquired resistance is observed in most patients treated with the two approved therapies for KRAS G12C mutant NSCLC, creating a significant opportunity for a therapy that re-sensitizes tumors to KRAS inhibition. Ribometrix’s data supports RBX-6610’s ability to deliver this mechanism in combination with the approved therapies. “ In less than three years, Ribometrix has advanced its eIF4E program from concept to a lead molecule that has just completed non-GLP toxicology studies,” stated Michael Solomon, CEO of Ribometrix, Inc. “ The program is a testament to Ribometrix drug discovery capabilities, and the commitment and professionalism of the Ribometrix team. We look forward to moving this program forward into the clinic in collaboration with a partner who shares our vision of its potential to benefit patients, via its novel mechanism of action.” “ These data further evidence the broad potential of eIF4E inhibition in multiple tumor types, particularly in combination as a re-sensitizing agent. We have further validated the mechanism of action and characterized the synergistic benefit of RBX-6610, our most advanced eIF4E inhibitor,” said Jessica Sorrentino, Ph.D., SVP of Translational Medicine. “ RBX-6610 is progressing towards the clinic and we see significant promise for its inclusion with the standard-of-care for both naïve and treatment resistant NSCLC patients, especially whose tumors have KRAS mutations.” Key highlights: RBX-6610 demonstrated consistent anti-proliferative effects in KRAS G12C mutant tumor cell lines, in both treatment-naïve lines and those with acquired resistance. Additionally, the combined in vitro effects of co-treatment of RBX-6610 with KRAS inhibitors were synergistic, such that the proliferative inhibitory effects were greater than the sum of either treatment alone. Mechanistically, this was due to an induction of apoptosis, specific to co-treatment in treatment-naïve tumor cells as well as cells resistant to KRAS inhibitors. This synergistic phenotype was observed with both approved KRAS inhibitors, as well as investigational RAS inhibitors. Daily oral RBX-6610 monotherapy led to substantial tumor growth inhibition in both treatment naïve and resistant KRAS G12C NSCLC xenograft model (75% vs 71% TGI, respectively), corroborating the anti-proliferative effects seen in vitro . In combination with sotorasib, an approved KRAS inhibitor, daily oral treatment with RBX-6610 led to significant tumor regression and improved survival in both the treatment-naïve and sotorasib resistant setting (34% and 49% mean tumor regression respectively). No toxicity was observed in up to 35 days of daily oral treatment across all cohorts. In vitro data demonstrated improved selectivity of cap dependent translation inhibition by RBX-6610 relative to other translation inhibitors including zotatifin, an eIF4A inhibitor. Ribometrix continues to generate data demonstrating the multi-tumor therapeutic potential of eIF4E inhibition. Studies in melanoma and breast cancer were presented at the Society for Melanoma Research and the San Antonio Breast Cancer Symposium in 2023, and data across melanoma, colorectal, breast, and non-small cell lung cancer were presented at the American Association for Cancer Research Annual Meeting in 2024. Data have repeatedly included support for both monotherapy and combination regimens as well as the potential to re-sensitize to standard-of-care treatments. The ESMO poster is now available to view on the “ Publications ” page of Ribometrix’s website. About eIF4E Eukaryotic translation initiation factor 4E (eIF4E) is a crucial regulatory component of mRNA translation and well-documented driver of oncogenesis. Clinically, eIF4E activity is elevated in many tumor indications and it is typically associated with poor prognosis. Targeting eIF4E has the potential to enhance anti-cancer activity when given in combination with standard-of-care. Additionally, eIF4E inhibition has the potential to overcome drug resistance and re-sensitize tumors to anti-cancer therapies. Based on substantial external and in-house data, Ribometrix is developing eIF4E inhibitors as a promising combination therapy approach and treatment for treatment-resistant tumors. About Ribometrix Ribometrix is a biotechnology company pioneering a completely new class of small molecule therapeutics that modulate RNA biology to treat human diseases. Ribometrix leverages its world-leading expertise in three-dimensional RNA structural analysis to identify novel small molecules that inhibit the production of disease-associated proteins. Ribometrix is advancing multiple internal programs including one targeting the oncogenic RNA-binding protein eIF4E and one directly targeting oncogenic c-MYC mRNA, and has established collaborations with Genentech, a member of the Roche Group, and Vertex Pharmaceuticals, which leverage Ribometrix’s discovery platform. Ribometrix is headquartered in Durham, North Carolina.

DURHAM, N.C.--( BUSINESS WIRE )-- Ribometrix , a biotechnology company developing small molecule therapeutics that modulate RNA biology, today announced the latest data from its eIF4E program will be presented at the European Society for Medical Oncology (ESMO) Congress, taking place in Barcelona, Spain, September 13-17. A poster presentation will review in vitro and in vivo studies of a small molecule eIF4E inhibitor, RBX-6610, as a potential treatment for non-small cell lung cancer (NSCLC), specifically NSCLC with KRAS G12C , a common mutation. Acquired resistance is observed in the majority of patients treated with the two approved therapies for KRAS G12C mutant NSCLC, creating a significant opportunity for a therapy that resensitizes tumors to these treatments. Ribometrix’s data supports RBX-6610’s ability to deliver this mechanism in combination with the approved therapies. “ The close relationship of eIF4E and KRAS signaling suggested the potential for eIF4E inhibition to restore tumor sensitivity to KRAS inhibition, and it is exciting to see this thesis borne out given the important medical need among patients with NSCLC,” said Jessica Sorrentino, Ph.D., SVP of Translational Medicine. “ We look forward to sharing our full dataset at ESMO 2024.” In vitro outcomes in the poster include: RBX-6610 monotherapy demonstrated consistent anti-proliferative effects in KRAS G12C mutant tumor cell lines, in both treatment-naïve lines and those with acquired resistance RBX-6610 combined with KRAS inhibitors resulted in a synergistic apoptotic induction in treatment-naïve tumor cells and re-sensitized resistant tumor cells to KRAS inhibitors. In vivo outcomes in the poster include: RBX-6610 monotherapy caused significant tumor growth inhibition RBX-6610 combined with a KRAS inhibitor resulted in significant tumor regression in a treatment-naïve model A further in vivo study reviewing RBX-6610’s ability to re-sensitize tumor cells to KRAS inhibition will be included in the final poster presentation. The presentation details are: Date : Sunday, September 15, 2024 Location : Dedicated poster area of Hall 6. Presentation number : 202P Title: eIF4E inhibition exhibits anti-tumor activity and re-sensitizes acquired resistant KRAS G12C NSCLC to KRAS inhibitors The ESMO poster will be available to view on the “ Publications ” page of Ribometrix’s website following the presentation. About eIF4E Eukaryotic translation initiation factor 4E (eIF4E) is a crucial regulatory component of mRNA translation and well-documented driver of oncogenesis. Clinically, eIF4E activity is elevated in many tumor indications and it is typically associated with poor prognosis. Targeting eIF4E has the potential to enhance anti-cancer activity when given in combination with standard-of-care. Additionally, eIF4E inhibition has the potential to overcome drug resistance and re-sensitize tumors to anti-cancer therapies. Based on substantial external and in-house data, Ribometrix is developing eIF4E inhibitors as a promising combination therapy approach and treatment for treatment-resistant tumors. About Ribometrix Ribometrix is a biotechnology company pioneering a completely new class of small molecule therapeutics that modulate RNA biology to treat human diseases. Ribometrix leverages its world-leading expertise in three-dimensional RNA structural analysis to identify novel small molecules that inhibit the production of disease-associated proteins. Ribometrix is advancing multiple internal programs including one targeting the oncogenic RNA-binding protein eIF4E and one directly targeting oncogenic c-MYC mRNA, and has established collaborations with Genentech, a member of the Roche Group, and Vertex Pharmaceuticals, which leverage Ribometrix’s discovery platform. Ribometrix is headquartered in Durham, North Carolina.