Broadly neutralizing SARS-CoV-2 peptidic knobs

摘要 靶向治疗药物的大小从非常小的有机分子(100‘S)到 以蛋白质为基础的分子，如单抗(约150,000 Da)。二硫键小分子 在包括植物和动物在内的许多物种中，多肽已经进化成具有理想的 药理特性包括高亲和力靶标结合，对蛋白酶的稳定性，热和 其他压力。这类多肽包括能够抑制酶、离子的“环肽”或“结蛋白”。 通道和GPCRs具有很高的效力，通常是蛇毒的主要活性成分 在许多捕食性生物中。我们发现了超长三分之一之间的收敛进化 一种特殊类型奶牛重链中的互补决定区(CDR H3s) 抗体和环肽/结蛋白多肽。我们可以在微生物体内生产这些“旋钮”多肽。 它们保留了亲本抗体的结合和效力特性。这些微小的 基于多肽的分子很小(~4-6 kDa)，高度稳定，可以与靶分子结合 亚纳分子KD。我们已经开发了一组病毒中和旋转肽 抗SARS-CoV-2，结合独特的表位，其中一些保持高亲和力 与各种SARS-CoV-2变种结合，包括最近的‘Delta’和‘Omicron’毒株。这个 高稳定性、高效性和直截了当的制造路径支持多种路径 给药，可能包括吸入或鼻腔给药，这可能非常重要 在当前或未来的冠状病毒大流行中进行预防或治疗。我们在这个项目中的目标 是进一步开发识别多肽结节结构域的技术，扩大我们的小组 针对SARS-CoV-2变种以及MERS-CoV和SARS-CoV等其他冠状病毒的旋钮- 了解它们结合的结构基础，并在体外和体内验证它们的活性。 这里确定的旋钮可能被用作单一疗法或联合疗法在 目前或新的冠状病毒大流行，并将是一个有价值的新的治疗类增加 对抗冠状病毒疾病的武器库。

Abstract Targeted therapeutic agents range in size from very small organic molecules (100’s of Da) to protein-based molecules like monoclonal antibodies (~150,000 Da). Small disulfide bonded peptides have evolved in many species, including plants and animals, to have ideal pharmacological properties including high affinity target binding, stability to proteases, heat and other stresses. Such peptides include “cyclotides” or “knottins” which can inhibit enzymes, ion channels, and GPCRs with high potency and are often the major active component of venoms of many predator organisms. We have uncovered convergent evolution between ultralong third complementary determining regions (CDR H3s) in the heavy chain of an unusual class of cow antibodies and cyclotide/knottin peptides. We can produce these “knob” peptides in microbial systems and they retain the binding and potency properties of the parent antibody. These tiny peptide-based molecules are small (~4-6 kDa), highly stable, and can bind targets at subnanomolar KD. We have already developed a panel of virus neutralizing knob peptides against SARS-CoV-2 which bind unique epitopes, and some of which maintain high affinity binding to various SARS-CoV-2 variants, including the recent ‘delta’ and ‘omicron’ strains. The high stability, potency, and straightforward manufacturing path enables multiple routes of administration, potentially including inhaled or intranasal delivery, which could be very important prophylactic or treatment in the current or future coronavirus pandemic. Our goals in this project are to further develop the technology to identify peptidic knob domains, expand our panel of knobs against SARS-CoV-2 variants and other coronaviruses like MERS-CoV and SARS-CoV- 1, understand the structural basis of their binding, and validate their activity in vitro and in vivo. The knobs identified here can potentially be used as monotherapy or combination therapy in the current or a new coronavirus pandemic, and will be a valuable new therapeutic class to add to the arsenal against coronavirus disease.