School of Life Sciences and Center of Novel Biomaterials, The Chinese University of Hong Kong, Hong Kong SAR 999077, China.
J Am Chem Soc. 2023 May 10;145(18):10249-10258. doi: 10.1021/jacs.3c01291. Epub 2023 Apr 26.
Macrocyclization has been touted as an effective strategy to enhance the in vivo stability and efficacy of protein therapeutics. Herein, we describe a scalable and robust system based on the endogenous biosynthesis of a noncanonical amino acid coupled to the pyrrolysine translational machinery for the generation of lasso-grafted proteins. The biosynthesis of the noncanonical amino acid d-Cys-ε-Lys was achieved by hijacking the pyrrolysine biosynthesis pathway, and then, its genetical incorporation into proteins was performed using an optimized PylRS/tRNA pair and cell line. This system was then applied to the structurally inspired cyclization of a 23-mer therapeutic P16 peptide engrafted on a fusion protein, resulting in near-complete cyclization of the target cyclic subunit in under 3 h. The resulting cyclic P16 peptide fusion protein possessed much higher CDK4 binding affinity than its linear counterpart. Furthermore, a bifunctional bicyclic protein harboring a cyclic cancer cell targeting RGD motif on the one end and the cyclic P16 peptide on the other is produced and shown to be a potent cell cycle arrestor with improved serum stability.
环化已被吹捧为一种增强蛋白质治疗剂体内稳定性和疗效的有效策略。在此，我们描述了一种基于非典型氨基酸的内源性生物合成与吡咯赖氨酸翻译机制偶联的可扩展且强大的系统，用于生成套索接枝蛋白。非典型氨基酸 d-Cys-ε-Lys 的生物合成是通过劫持吡咯赖氨酸生物合成途径来实现的，然后使用优化的 PylRS/tRNA 对和细胞系将其遗传掺入蛋白质中。然后将该系统应用于受结构启发的 23 肽治疗性 P16 肽接枝融合蛋白的环化，结果在不到 3 小时内目标环状亚基几乎完全环化。所得的环状 P16 肽融合蛋白与线性对应物相比具有更高的 CDK4 结合亲和力。此外，还生产了一种具有双功能的双环蛋白，其一端带有环状癌细胞靶向 RGD 基序，另一端带有环状 P16 肽，并且表现出改善的血清稳定性的强效细胞周期抑制剂。
