中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2013年
5期
363-366
,共4页
分枝杆菌,结核%蛋白酶体抑制剂
分枝桿菌,結覈%蛋白酶體抑製劑
분지간균,결핵%단백매체억제제
Mycobacterium tuberculosis%Proteasome inhibitors
目的 建立 MTB蛋白酶体抑制剂体外筛选模型.方法 根据荧光底物N-琥珀酰基-亮氨酸-亮氨酸-缬氨酸-酪氨酸-7-氨基-4-甲基香豆素(S-LLVY-AMC)在蛋白酶体作用下发生水解,释放出具有荧光的7-氨基-4-甲基香豆素(AMC),且荧光值与蛋白酶体活性呈正比的原理,建立蛋白酶体抑制剂体外筛选模型,并对该体系中影响蛋白酶体活性的反应时间及十二烷基磺酸钠(SDS)、蛋白和底物浓度进行优化,利用已知蛋白酶体抑制剂MG132及8种天然产物对该模型进行验证.对荧光值的影响因素进行直线相关与回归分析.结果 MTB蛋白酶体抑制剂体外筛选模型中SDS为0.34 mg/L,蛋白浓度为25 mg/L,底物S-LLVY-AMC浓度为64 μmol/L,反应时间为30 min.利用MG132及药物A～H天然产物对MTB蛋白酶体筛选模型进行验证,经计算得出MG132半抑制浓度为49 μmol/L.在终浓度为200 μmol/L时,8种天然产物对MTB蛋白酶体的抑制率分别为0.0％、3.1％、7.8％、45.2％、52.4％、69.5％、69.6％和88.7％.证实该模型具有重复性及可靠性.结论 本研究成功建立MTB蛋白酶体抑制剂体外筛选模型,该模型为快速、较大规模筛选MTB蛋白酶体抑制剂提供良好的技术平台.
目的 建立 MTB蛋白酶體抑製劑體外篩選模型.方法 根據熒光底物N-琥珀酰基-亮氨痠-亮氨痠-纈氨痠-酪氨痠-7-氨基-4-甲基香豆素(S-LLVY-AMC)在蛋白酶體作用下髮生水解,釋放齣具有熒光的7-氨基-4-甲基香豆素(AMC),且熒光值與蛋白酶體活性呈正比的原理,建立蛋白酶體抑製劑體外篩選模型,併對該體繫中影響蛋白酶體活性的反應時間及十二烷基磺痠鈉(SDS)、蛋白和底物濃度進行優化,利用已知蛋白酶體抑製劑MG132及8種天然產物對該模型進行驗證.對熒光值的影響因素進行直線相關與迴歸分析.結果 MTB蛋白酶體抑製劑體外篩選模型中SDS為0.34 mg/L,蛋白濃度為25 mg/L,底物S-LLVY-AMC濃度為64 μmol/L,反應時間為30 min.利用MG132及藥物A～H天然產物對MTB蛋白酶體篩選模型進行驗證,經計算得齣MG132半抑製濃度為49 μmol/L.在終濃度為200 μmol/L時,8種天然產物對MTB蛋白酶體的抑製率分彆為0.0％、3.1％、7.8％、45.2％、52.4％、69.5％、69.6％和88.7％.證實該模型具有重複性及可靠性.結論 本研究成功建立MTB蛋白酶體抑製劑體外篩選模型,該模型為快速、較大規模篩選MTB蛋白酶體抑製劑提供良好的技術平檯.
목적 건립 MTB단백매체억제제체외사선모형.방법 근거형광저물N-호박선기-량안산-량안산-힐안산-락안산-7-안기-4-갑기향두소(S-LLVY-AMC)재단백매체작용하발생수해,석방출구유형광적7-안기-4-갑기향두소(AMC),차형광치여단백매체활성정정비적원리,건립단백매체억제제체외사선모형,병대해체계중영향단백매체활성적반응시간급십이완기광산납(SDS)、단백화저물농도진행우화,이용이지단백매체억제제MG132급8충천연산물대해모형진행험증.대형광치적영향인소진행직선상관여회귀분석.결과 MTB단백매체억제제체외사선모형중SDS위0.34 mg/L,단백농도위25 mg/L,저물S-LLVY-AMC농도위64 μmol/L,반응시간위30 min.이용MG132급약물A～H천연산물대MTB단백매체사선모형진행험증,경계산득출MG132반억제농도위49 μmol/L.재종농도위200 μmol/L시,8충천연산물대MTB단백매체적억제솔분별위0.0％、3.1％、7.8％、45.2％、52.4％、69.5％、69.6％화88.7％.증실해모형구유중복성급가고성.결론 본연구성공건립MTB단백매체억제제체외사선모형,해모형위쾌속、교대규모사선MTB단백매체억제제제공량호적기술평태.
Objective To establish a model for screening Mycobacterium tuberculosis (M.tuberculosis) proteasome inhibitors in vitro.Methods The proteasome inhibitor in vitro screening model was established according to the principle that the fluorescent substrate Suc-Leu-Leu-Val-AMC (S-LLVY-AMC)was hydrolyzed by the proteasome and released AMC (7-amino-4-methylcoumarin) which was fluorescent,and the fluorescence value was proportional to the proteasome activity.Conditions which could affect proteasome activity including the time of the reaction,concentration of proteasome,or SDS,were evaluated.The model was validated by using the known proteasome inhibitor MG132 and 8 natural products.Linear correlation and regression analysis were performed by the factors affecting fluorescence values.Results In the model for screening M.tuberculosis proteasome inhibitors in vitro,the reaction system consisted of 0.34mg/L SDS,25 mng/L protein and 64 μmol/L S-LLVT-AMC,and the reaction time was 30 min.By using MG132 and natural compounds A-H to validate the MTB proteasome screening model,the calculated IC50of MG132 was 49 μmol/L.In a final concentration of 200 μmol/L,the inhibition rates of 8 drugs on MTB proteasome were 0.0％,3.1％,7.8％,45.2％,52.4％,69.5％,69.6％,88.7％ respectively,confirming that the model was repeatable and reliable.Conclusions In this study,we established a model for screening M.tuberculosis proteasome inhibitors in vitro,which could provide a technical platform for fast and large scale screening of mycobacterial proteasome inhibitors.
