Zhao Ming, Wan Zhu-li, Whittaker Linda, Xu Bin, Phillips Nelson B, Katsoyannis Panayotis G, Ismail-Beigi Faramarz, Whittaker Jonathan, Weiss Michael A
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 2009 Nov 13;284(46):32178-87. doi: 10.1074/jbc.M109.028399. Epub 2009 Sep 22.
Insulin binds with high affinity to the insulin receptor (IR) and with low affinity to the type 1 insulin-like growth factor (IGF) receptor (IGFR). Such cross-binding, which reflects homologies within the insulin-IGF signaling system, is of clinical interest in relation to the association between hyperinsulinemia and colorectal cancer. Here, we employ nonstandard mutagenesis to design an insulin analog with enhanced affinity for the IR but reduced affinity for the IGFR. Unnatural amino acids were introduced by chemical synthesis at the N- and C-capping positions of a recognition alpha-helix (residues A1 and A8). These sites adjoin the hormone-receptor interface as indicated by photocross-linking studies. Specificity is enhanced more than 3-fold on the following: (i) substitution of Gly(A1) by D-Ala or D-Leu, and (ii) substitution of Thr(A8) by diaminobutyric acid (Dab). The crystal structure of [D-Ala(A1),Dab(A8)]insulin, as determined within a T(6) zinc hexamer to a resolution of 1.35 A, is essentially identical to that of human insulin. The nonstandard side chains project into solvent at the edge of a conserved receptor-binding surface shared by insulin and IGF-I. Our results demonstrate that modifications at this edge discriminate between IR and IGFR. Because hyperinsulinemia is typically characterized by a 3-fold increase in integrated postprandial insulin concentrations, we envisage that such insulin analogs may facilitate studies of the initiation and progression of cancer in animal models. Future development of clinical analogs lacking significant IGFR cross-binding may enhance the safety of insulin replacement therapy in patients with type 2 diabetes mellitus at increased risk of colorectal cancer.
胰岛素与胰岛素受体（IR）具有高亲和力，与1型胰岛素样生长因子（IGF）受体（IGFR）具有低亲和力。这种交叉结合反映了胰岛素 - IGF信号系统内的同源性，在高胰岛素血症与结直肠癌的关联方面具有临床意义。在此，我们采用非标准诱变设计一种对IR亲和力增强但对IGFR亲和力降低的胰岛素类似物。通过化学合成在识别α-螺旋（残基A1和A8）的N端和C端封端位置引入非天然氨基酸。光交联研究表明，这些位点毗邻激素 - 受体界面。在以下方面特异性提高了3倍以上：（i）用D - 丙氨酸或D - 亮氨酸取代Gly（A1），以及（ii）用二氨基丁酸（Dab）取代Thr（A8）。在六聚体锌（T6）中测定的[D - Ala（A1），Dab（A8）]胰岛素的晶体结构分辨率为1.35 Å，与人类胰岛素基本相同。非标准侧链在胰岛素和IGF - I共有的保守受体结合表面边缘伸向溶剂。我们的结果表明，在这个边缘的修饰可以区分IR和IGFR。由于高胰岛素血症的典型特征是餐后胰岛素浓度积分增加3倍，我们设想这种胰岛素类似物可能有助于在动物模型中研究癌症的发生和发展。未来开发缺乏显著IGFR交叉结合的临床类似物可能会提高结直肠癌风险增加的2型糖尿病患者胰岛素替代治疗的安全性。
