促红细胞生成素B螺旋表面肽的代谢稳定性结构修饰:设计、合成及其提高的肾保护作用

Metabolically stabilized structural modification on the helix B surface peptide of erythropoietin: Design, synthesis and improved renoprotective effect

肾脏缺血缺氧以及再灌注过程都将导致肾小管上皮细胞凋亡,使肾功能严重受损.肾脏的缺血再灌注损伤是移植肾功能延迟恢复的主要原因并能诱导急慢性排斥,影响肾存活率.近年来发现,衍生于促红细胞生成素(EPO)的B螺旋亚基亲水表面序列的肽链(HBSP),对肾脏缺血再灌注损伤具有显著的保护作用,但其在体内极短的半衰期(约2min)极大地限制了它的临床应用.因此,本研究采用构象约束、全D-构型氨基酸替换和N-端封闭策略,设计了3种类型的EPOB螺旋表面肽衍生物,旨在提高其代谢稳定性环肽的设计采用了对氧化还原稳定的硫醚键和相对刚性的亚砜键两种环合方式.在多肽的合成上,采用微波辅助多肽自动合成和手工合成两种模式有机结合;优化了硫醚环合工艺,应用微波加热进行硫醚环肽的合成,大大提高了产率和效率;利用圆二色(CD)谱确定了亚砜环肽的相对构型.活性实验表明,相对于线性母肽HBSP,本文设计合成的代谢稳定衍生肽对大/小鼠肾脏缺血再灌注损伤均有显著提高的保护作用,且硫醚和R-构型亚砜环肽的肾脏保护活性强于S-构型亚砜环肽.而且,环化确实提高了功能肽的血浆稳定性.因此,本文合成的硫醚环肽一周一次注射剂量等效于线性肽HBSP一日三次剂量对小鼠肾损伤的保护作用.

Kidney ischemia reperfusion （IR） injury is a major cause of delayed graft function and can increase the risk of allograft rejection, affecting both short- and long-term graft survivals. It has been recently shown that helix B surface peptide （HBSP）, an 11-amino acid long sequence derived from the aqueous surface of the helix B domain of erythropoietin, has powerful tissue protective function in various organs subjected to IR injury. However, the 2 minplasma half-life of HBSP restricts its application in vivo. In this study, conformationally constraining, all D-amino acid replacement and N-capping strategies were employed to modify the structure and thus improved the metabolic stability of the linear peptide HBSP. The redox-stable thioether and relatively rigid sulfoxide were chosen as the linkage to tether the 11-amino acid binding motif. For the synthesis of these peptide derivatives, microwave peptide synthesizer and manual solid peptide synthesis were combined to improve the efficiency. Especially, microwave assisted macrocyclization greatly facilitated the production of the thioether-cyclized peptide. The configuration of the chiral sulfoxide linkage was identified by CD spectra. Significantly, the renal functional assay in a murine kidney IR model indicated that these metabolically stabilized derivatives displayed improved renal protective effect compared to the linear parent peptide. Furthermore, the thioether-cyclized peptide was superior to the sulfoxide-cyclized peptide with respect to reducing the serum creatinine level. Therefore, the TE-CHBP with one dose in one week maintained equal renoprotective effect to the HBSP with 3 doses in one week in murine kidney ischemia reperfusion （IR） model.