γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ^2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (M^pro). Two kinds of cyclic γ^2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ¹) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ²), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent M^pro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ¹ at the fourth position, manifests a 5.2 nM dissociation constant. An M^pro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ¹ interacts with the S1′ catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and M^pro enabled production of a variant with a 5-fold increase in potency.

γ-氨基酸在天然产物的生物活性中可发挥重要作用；然而，将γ-氨基酸核糖体掺入肽中具有挑战性。^{在此，我们报告了使用含有环状 γ 2,4-}氨基酸的非规范肽库的筛选活动如何导致发现 SARS-CoV-2 主要蛋白酶 (M ^pro ) 的非常有效的抑制剂。将两种环状γ ^2,4 -氨基酸，顺式-3-氨基环丁烷甲酸(γ ¹ )和(1 R ,3 S )-3-氨基环戊烷甲酸(γ ² )，通过核糖体引入硫醚库中-大环肽。一种有效的 M ^pro合成物抑制剂（半最大抑制浓度 = 50 nM）GM4，包含 13 个残基，第四位为 γ ^{1 ，解离常数为 5.2 nM。}M ^pro :GM4 复合晶体结构揭示了完整的抑制剂跨越底物结合裂缝。γ ¹与 S1' 催化亚位点相互作用，与丙氨酸取代的变体相比，使蛋白水解稳定性提高 12 倍。了解 GM4 和 M ^pro之间的相互作用使得能够生产效力增加 5 倍的变体。