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

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_(12)-CPI-1的8 h胰蛋白酶降解率显著降低(P<0.05,P<0.01)。小鼠实验结果表明,2×LD_(50) BoNT/A与CPI非长修饰衍生物共孵育30 min后ip给予小鼠,与毒素组相比,毒素+CPI-1组小鼠72 h内存活率无明显差异;毒素+衍生物CPI-1-L,CPI-1-W,CPI-1-Y,CPI-1-R和CPI-1-K组小鼠72 h内存活率明显提高(P<0.05),且与毒素+CPI-1组相比也均明显提高(P<0.05);2.5×LD_(50) BoNT/A与CPI长链修饰衍生物共孵育30 min后ip给予小鼠,与毒素组相比,毒素+CPI-1组及毒素+衍生物CPI-1-LK(stearic)GG、C_(12)-CPI-1、C_(14)-CPI-1和C_(16)-CPI-1组小鼠72 h内存活率明显提高(P<0.05),毒素+衍生物CPI-1-K(stearic)GG,CPI-1-LGK(stearic)GG及C_(8)-CPI-1组无明显差异;与毒素+CPI-1组相比,毒素+衍生物各组均无明显差异。2×LD_(50) BoNT/A攻毒2和6 h后给予CPI衍生物进行解救,与毒素组相比,毒素+C_(12)-CPI组小鼠72 h内存活率明显提高(P<0.05),毒素+CPI-1及其余衍生物组无明显差异;与毒素+CPI-1相比,毒素+各衍生物组小鼠72 h内存活率均无明显差异。结论CPI-1 C端引入疏水或碱性氨基酸可提高对轻链的抑制活性,C端脂肪链修饰衍生物抗酶解能力增强,N端月桂酸修饰衍生物在小鼠体内具有明显的抗BoNT/A活性。

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 at the C-terminal of CPI-1 resulted in a 3.5-0.8-fold increase in the inhibitory activity(IC_(50)=0.06-0.15μmol·L^(-1))compared to CPI-1[IC_(50)=(0.27±0.02)μmol·L^(-1)](P<0.01),but the introduction of acidic amino acid glutamic(7)led to a 78.6%decrease in the potency[IC_(50)=(1.26±0.28)μmol·L^(-1)](P<0.01).After the introduction of fatty acid at the N-or C-terminal of CPI-1,the potency(IC_(50)>1.36μmol·L^(-1))was decreased by at least 80%(P<0.01).The anti-enzymatic activity tests showed that after the introduction of fatty acid at the N-or C-terminal of CPI-1,the trypsin degradation rate of the CPI-1-K(stearic)GG and C_(12)-CPI-1 was significantly decreased at 8 h(P<0.05,P<0.01).Detoxification experiments showed that after co-injection of 2×LD_(50) BoNT/A with 5 mg·kg^(-1) inhibitor the survival rate of mice in CPI-1-L,CPI-1-W,CPI-1-Y,CPI-1-R,and CPI-1-K groups was significantly increased in 72 h compared to the blank control group(P<0.05),but not in the CPI-1 group and the survival rate of mice was more significantly increased than in the CPI-1 group(P<0.05).After co-injection(ip)of 2.5×LD_(50) BoNT/A with 5 mg·kg^(-1) inhibitor,the survival rate of mice in CPI-1,CPI-1-LK(stearic)GG,C_(12)-CPI-1,C_(14)-CPI-1 and C_(16)-CPI-1 groups was also more significantly increased than in the blank control group in 72 h(P<0.05)rather than in CPI-1-K(stearic)GG,CPI-1-LGK(stearic)GG and C_(8)-CPI-1 groups.The survival rate of mice in other groups was not siginificantly different from that of the CPI-1 group.2 and 6 h after the injection(ip)of BoNT/A(2×LD_(50))and treatment with the 5 mg·kg^(-1) inhibitor,the survival rate of mice in the C_(12)-CPI-1 group was more significantly increased than in the blank control group in 72 h(P<0.05),but not in other groups.The survival rate of mice in other groups was not significantly different from that of the CPI-1 group.CONCLUSION The introduction of hydrophobic amino acids and basic amino acids at the C-terminual of CPI-1 results in an increase in potency.The addition of fatty acid at C-or N-terminal enhances the anti-enzymatic activity of CPI-1.The CPI-1 derivative modified by dodecanoic acid displays apparent detoxification activity to BoNT/A.