长效胰岛素的分子设计研究

STUDIES ON PROLONGED ACTING INSULIN MOLECULAR DESIGN

以工程化制备由分子内在因素制导的可溶性长效胰岛素为目标,提出以“增加次级键,稳化寡聚体”原理,用以减缓注射进入体内的胰岛素六聚体的解聚速率,形成能持续供应胰岛素单体的“库”,从而获得长效作用。据此以精确的胰岛素三维结构为基础,进行了分子设计和计算机模拟,提出了对胰岛素的B链c端和N端修饰产生长效性的分子改造方案,重点分析了B2Lys/Arg和B31Lys/Arg胰岛素。用化学半合成制备的上列二种胰岛素,生物学测试表明确有长效性。2埃分辨率B31-Arg人胰岛素晶体结构的测定显示了Arg-B31与相邻分子Glu-B21间存在一对附加氢键作为长效效应的主要结构基础,证实分子设计思想的基本正确性。

In order to prepare soluble and prolonged-acting insulin derivatives which is mainly directed by intramolecular interactions, a principle is proposed. That is, making some additional non-covalent bonds between monomers of insulin oligomer through residue modification so as to slow down the dissociation rate of insulin hexamer injected into the body and produce the protraction from a depot-effect. On the basis of this idea and the fine structure of insulin, molecular design and computer modelling have been undertaken which provided some rational schemes for site-directed mutagenesis mainly dealing with N-and C-terminal residues of B chain. Among others, B2-Lys/Arg and B31-Lys/Arg substitutions were analysed in some detail and semisynthseized chemically as target samples. Biological tests in rabbits showed that they really both exhibited protracted glucose lowering effects. Furthermore an X-ray structure analysis of B31-Arg human insulin has also been completed at 2 A resolution and showed a pair of additional charged hydrogen bond formed by Arg-B31 and Glu-B21 of the neighbouring monomer in a dimer, that should be the structural basis of such prolonged action. This demonstrates the rationality of our idea for the molecular design.