A型肉毒毒素轻链肽类抑制剂CPI-1衍生物的合成及解毒活性评价  

Synthesis and detoxification activities evaluation of derivatives of botulinum toxin A light chain inhibitor CPI-1

作　　者：徐书静 沈锦涛 刘佳 黄悦 余硕 余云舟 孙黔云 戴秋云[2] XU Shujing;SHEN Jintao;LIU Jia;HUANG Yue;YU Shuo;YU Yunzhou;SUN Qianyun;DAI Qiuyun(Institute of Pharmacy,Guizhou Medical University,Guiyang 550000,China;Institute of Biotech-nology,Academy of Military Medical Sciences,Beijing 100071,China;The Key Laboratory of Chemistry for Natural Products,Guizhou Province and Chinese Academy of Sciences,Guiyang 550014,China)

机构地区：[1]贵州医科大学药学院,贵州贵阳550000 [2]军事科学院军事医学研究院生物工程研究所,北京100071 [3]贵州省中国科学院天然产物化学重点实验室,贵州贵阳550014

出　　处：《中国药理学与毒理学杂志》2023年第8期592-600,共9页Chinese Journal of Pharmacology and Toxicology

基　　金：国家重点研发计划(2017YFC1200905)。

摘　　要：目的合成多肽CPI-1的N端或C端衍生物并进行活性评价,以获得活性更高、药动学性质更好的A型肉毒毒素(BoNT/A)轻链抑制剂。方法采用固相方法合成CPI-1 C端增加不同氨基酸的非长链衍生物〔CPI-1-L(1),CPI-I-GL(2),CPI-1-W(3),CPI-1-Y(4),CPI-1-R(5),CPI-1-K(6)和CPI-1-E(7)〕及C端或N端引入长链脂肪酸的长链衍生物〔CPI-1-K(stearic)GG(8),CPI-1-LK(stearic)GG(9),CPI-1-LGK(stearic)GG(10),C_(8)-CPI-1(11),C_(12)-CPI-1(12),C_(14)-CPI-1(13)和C_(16)-CPI-1(14)〕。线性肽CPI-1及非长链修饰衍生物完成固相合成后,脱除保护基后在NH_(4)HCO_(3)溶液中氧化折叠成环状目标肽;CPI-1长链修饰衍生物线性肽完成固相合成后,在树脂上直接碘氧化关环,然后裂解。各粗肽经高效液相色谱法(HPLC)纯化后得到目标肽。采用荧光共振能量转移法测定目标多肽对轻链片段LC424的抑制活性,HPLC测定多肽对胰蛋白酶酶解的稳定性。小鼠随机分为毒素组、毒素+CPI-1组及毒素+CPI-1衍生物组,①将生理盐水、5 mg·kg^(-1) CPI-1或CPI-1衍生物分别与2×LD_(50)(或2.5×LD_(50))BoNT/A孵育后ip给予小鼠,测定72 h内小鼠存活率;②以2×LD_(50) BoNT/A攻毒2和6 h后,分别给予生理盐水和5 mg·kg^(-1) CPI-1或CPI-1衍生物进行解救,观察72 h内小鼠存活率。结果共合成14个CPI-1衍生物,其中7个非长链修饰衍生物,7个长链修饰衍生物,经质谱鉴定相对分子质量正确。LC424抑制实验结果表明,CPI-1 C端引入疏水氨基酸亮氨酸(CPI-1-L)、色氨酸(CPI-1-W)、酪氨酸(CPI-1-Y)及碱性氨基酸精氨酸(CPI-1-R)后,其对轻链的抑制活性(IC_(50)=0.06~0.15μmol·L^(-1))较CPI-1提高3.5~0.8倍(P<0.01),引入酸性氨基酸谷氨酸(Glu)(CPI-1-E)抑制活性(IC_(50)=1.26±0.28μmol·L^(-1))降低78.6%(P<0.01);C端或N端引入长链脂肪酸修饰后抑制活性(IC_(50)>1.36μmol·L^(-1))下降>80%(P<0.01)。酶降解稳定性实验结果表明,C端或N端引入长链脂肪酸修饰后,CPI-1-K(stearic)GG和C_OBJECTIVE To synthesize and evaluate N-terminal and C-terminal derivatives of CPI-1 in order to obtain light chain inhibitors with better activity and good pharmacokinetic performance.METHODS Non-long-chain derivatives modified by different amino acids at the C-terminal of CPI-1[CPI-1-L(1),CPI-I-GL(2),CPI-1-W(3),CPI-1-Y(4),CPI-1-R(5),CPI-1-K(6),CPI-1-E(7)]and long-chain derivatives modified by long-chain fatty acids at its C-or N-terminal[CPI-1-K(stearic)GG(8),CPI-1-LK(Stearic)GG(9),CPI-1-LGK(stearic)GG(10),C_(8)-CPI-1(11),C_(12)-CPI-1(12),C_(14)-CPI-1(13),C_(16)-CPI-1(14)]were synthesized using solid phase methods.After being deprotected,the linear peptide was folded into the cyclic target peptide in NH_(4)HCO_(3) buffer.The long-chain modified CPI-1 derivatives were oxidized on resin to form disulfide bonds before being cleaved.All crude peptides were purified by reversed phase-high pressure liquid chromatography(RP-HPLC).The inhibitory activities of peptides against BoNT/A light chain LC424(1-424)were determined by fluorescence resonance energy transfer assays,while the stability of enzymatic hydrolysis of peptides by trypsin was determined by RP-HPLC.Mice were randomly divided into the saline group,CPI-1 group and the CPI-1 derivative groups.2×LD_(50)(2.5×LD_(50))BoNT/A was co-incubated with saline,(5 mg·kg^(-1))CPI-1 or CPI-1 derivatives before being intraperitoneally(ip)injected into mice.The survival rates of mice were recorded for 72 h.2 and 6 h after ip injection of 2×LD_(50) BoNT/A,the mice were treated with saline,5 mg·kg^(-1) CPI-1 or CPI-1 derivatives before the survival rates of mice were recorded for 72 h.RESULTS Fourteen CPI-1 derivatives including 7 non-long-chain and 7 long-chain ones were synthesized.The relative molecular mass of all derivatives was consistent with the theoretical molecular mass by mass spectrometry.The results of inhibitory activities in vitro showed that the introduction of hydrophobic and basic amino acids[such as leucine(1),tryptophane(3),tyrosine(4)and arginine(5)]of

关 键 词：A型肉毒毒素 轻链抑制剂 CPI-衍生物 抑制活性 稳定性 

分 类 号：R965[医药卫生—药理学]