The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity, yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here, we have developed covalent nanobodies capable of binding with SARS-CoV-2 irreversibly via a proximity-enabled reactive therapeutic (PERx) mechanism. A latent bioreactive amino acid (FFY) was designed and genetically encoded into nanobodies to accelerate the PERx reaction rate. Compared with the noncovalent wild-type nanobody, the FFY-incorporated covalent nanobodies neutralized both wild-type SARS-CoV-2 and its Alpha, Delta, Epsilon, Lambda, and Omicron variants with drastically higher potency. This PERx-enabled covalent-nanobody strategy and the related insights into increased potency can be valuable to developing effective therapeutics for various viral infections.

持久的 COVID-19 大流行和不断增加的 SARS-CoV-2 变种需要有效的药物进行预防和治疗。基于蛋白质的生物制剂具有很高的特异性，但它们的非共价相互作用常常导致药物解离和不完全抑制。在这里，我们开发了能够通过邻近反应治疗 (PERx) 机制与 SARS-CoV-2 不可逆结合的共价纳米抗体。设计了一种潜在生物反应性氨基酸 (FFY)，并将其基因编码到纳米抗体中，以加速 PERx 反应速率。与非共价野生型纳米抗体相比，掺入 FFY 的共价纳米抗体能够中和野生型 SARS-CoV-2 及其 Alpha、Delta、Epsilon、Lambda 和 Omicron 变体，且效力显着提高。这种基于 PERx 的共价纳米抗体策略以及对增强效力的相关见解对于开发针对各种病毒感染的有效疗法非常有价值。