A型肉毒毒素轻链抑制剂CPI-1衍生物的合成及活性评价

Syntheses and activity evaluation of derivates of botulinum toxin a light chain inhibitor CPI-1

目的在A型肉毒毒素轻链多肽抑制剂CPI-1引入穿膜序列,提高其抑制活性。方法基于CPI-1序列,在N端或C端增加穿膜序列Angiopep-2(Ang)或多聚精氨酸(R8),固相合成线性肽,空气氧化折叠形成目标产物,经RP-HPLC纯化得纯肽。表达A型肉毒毒素轻链截短体LC424,合成含荧光基团与淬灭基团的肉毒毒素轻链水解底物SNAPtide,采用荧光共振能量转移法(FRET)测定各多肽对肉毒毒素轻链的抑制活性,小鼠攻毒测定各肽体内活性。结果将Ang连接于CPI-1的N端或C端后,Ang-CPI-1、Ang-GG-CPI-1、CPI-1-Ang以及CPI-1-GG-Ang的IC_(50)值分别为148. 2、251. 9、88. 1、90. 9 nmol/L,相比于CPI-1(IC_(50)=411. 9 nmol/L)活性提升1～3倍;多聚精氨酸接入CPI-1的N端后导致抑制活性丧失。结论在CPI-1的N端和C端增加Ang肽能提升其对A型肉毒毒素轻链抑制活性,但不能增加体内活性。

Objective To increase the inhibitory activity of the botulinum neurotoxin A(Bo NT/A)light chain inhibitor CPI-1 by introducing cell-penetrating peptides.Methods The transmembrane sequence Angiopep-2(Ang)or polyarginine(n=8)was introduced at N-or C-terminus of CPI-1,the linear peptides were synthesized using solid phase methods and were folded by air oxidation,while the aim peptides were purified by RP-HPLC.Truncated Bo NT/A light chain LC424 was expressed and its hydrolytic substrate SNAPtide containing fluorescent group and quenching group was also synthesized.The inhibitory activities of peptides to LC424 were determined by fluorescence resonance energy transfer(FRET)assays and in vivo detoxification activities were determined in mice.Results After the introduction of Angiopep-2 at the N-or C-terminus of CPI-1,the IC50 values of Ang-CPI-1,Ang-GG-CPI-1,CPI-1-Ang and CPI-1-GG-Ang were 148.2,251.9,88.1 and90.9 nmol/L,respectively,and were 1-3 times higher than that of CPI-1(411.9 nmol/L).The introduction of polyarginine at the N-terminus of CPI-1 resulted in the loss of inhibitory activity.Conclusion The introduction of Ang at the N-or C-terminus of CPI-1 increases its inhibitory activity to Bo NT/A light chain,but cannot improve the in vivo detoxification activity.