Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes.
Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes.
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DOI：
10.1371/journal.pone.0049345
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Xu J
中科院分区：
文献类型：
--
作者：
Hecht R;Li YS;Sun J;Belouski E;Hall M;Hager T;Yie J;Wang W;Winters D;Smith S;Spahr C;Tam LT;Shen Z;Stanislaus S;Chinookoswong N;Lau Y;Sickmier A;Michaels ML;Boone T;Véniant MM;Xu J
成纤维细胞生长因子21（FGF 21）是一种很有前途的治疗2型糖尿病的候选药物。然而，野生型天然FGF 21的使用由于若干限制而具有挑战性。其中包括其半衰期短、对体内蛋白水解降解的敏感性和体外聚集的倾向。我们在这里描述了一种基于理性的蛋白质工程方法，以产生一种有效的长效FGF 21类似物，具有改善的抗蛋白水解和聚集性。通过连接肽将人IgG 1的Fc结构域融合到人成熟FGF 21的N-末端，构建重组Fc-FGF 21融合蛋白。确定位于N-末端的Fc比C-末端的Fc优越上级，因为N-末端Fc融合体保留了与天然FGF 21相似的βKlotho结合亲和力和体外和体内效力。在FGF 21中引入了两个特定的点突变。在位置98处的亮氨酸至精氨酸的取代（L98 R）在高浓度和升高的温度下抑制FGF 21聚集。在位置171（P171 G）处的脯氨酸至甘氨酸的置换消除了在小鼠和食蟹猴中鉴定的FGF 21的位点特异性蛋白水解切割。衍生的Fc-FGF 21（RG）分子表现出显著改善的循环半衰期，同时保持与野生型蛋白相似的体外活性。与天然FGF 21或Fc-FGF 21野生型的1-2小时相比，Fc-FGF 21（RG）的半衰期在小鼠中为11小时，在猴中为30小时。在糖尿病小鼠中单次施用Fc-FGF 21（RG）导致血糖水平和体重增加持续降低长达5-7天，而FGF 21或Fc-FGF 21的功效仅持续1天。总之，我们设计了一种强效且有效的长效FGF 21类似物，其具有有利的药学性质，可用于潜在的临床开发。
Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.
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DOI：
10.1073/pnas.0701600104
发表时间：
2007-05-01
影响因子：
11.1
作者：
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