人促红细胞生成素小肽模拟物研究
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摘要
本文以分子动力学模拟方法和分子力学理论为基础,构建了自有的人促红细胞生成素(Erythropoietin,EPO)模拟肽(Erythropoietin Mimetic peptide,EMP)与其受体(Erythropoietin Receptor,EpoR)结合的动力学模型。通过对该模型的结构研究和理论分析,对EMP与EpoR结合机制提出了新的理论解释。以该理论为指导,设计合成了新的一系列EPO模拟肽(Mimetic Binding Peptides,MBPs)。经过对MBPs活性的实验检验,验证了理论和模型的准确性,并得到了具有较好活性的MBPs分子。
以人EpoR与EPO、EMP的复合物X-光衍射晶体结构(PDB ID:1CN4,1EBP等)为模板,利用该复合结构的X-光衍射结构分析数据,用分子力学和分子动力学方法,建立了EMP与EpoR结合的分子动力学模型,并对这个模型中的配体与受体相互作用机制进行了理论研究。
在对模型的结构研究和模拟计算中发现,静电相互作用是配体与受体相互作用的主要作用力。不同配体分子的空间结构和与受体的不同结合方式,会直接影响二聚后两EPO受体分子间夹角和距离,进而产生不同的活性作用——激活剂或者拮抗剂。
本文在对此模型的结构分析和配体与受体结合机制研究的理论基础上,设计了一系列新的EPO小肽类模拟物,我们将其命名为MBPs。使用电荷和疏水性质更强的部分氨基酸来取代原有的EMP中的相应氨基酸。经过计算分析,这些新的模拟肽MBPs在理论上应具有更好的结合和激活活性。
为了从实验上验证设计后的MBPs的结合与聚合活性,我们利用荧光标记法以及微孔板荧光技术建立了EPO模拟物的体外结合与聚合活性检测方法。
由活性检测所得到的实验结果,与理论计算和设计相比较,验证了我们建立的模型的可靠性及计算方法的合理性。为小肽类药物设计和开发开拓了一个新的设计思路和实践方法。
Activation of transmembrane receptors for cytokines occurs when oligomerization of receptor chains is induced by binding of a protein ligand (cytokine) to a specific ligand-binding domain on the receptor. The resulting clustering of tyrosine kinase domains on the cytoplasmic side of the receptor initiates signal transduction events. Receptors may also be activated by bivalent receptor antibodies and by dimeric peptides that interact with the ligand binding domain. Thus activation of receptors by small,peptidyl molecules might make it possible to develop cytokine mimetics that may replace natural counterparts that are now on the market for therapy. This is illustrated here for erythropoietin (EPO). EPO,used for stimulating production of red blood cells,is a major product in biotechnology with world-wide sales of $2.6 billion already in 1995. Thus there has been an increasing interest in molecular mimicry with EPO potency for obviously huge commercial
以人EpoR与EPO、EMP的复合物X-光衍射晶体结构(PDB ID:1CN4,1EBP等)为模板,利用该复合结构的X-光衍射结构分析数据,用分子力学和分子动力学方法,建立了EMP与EpoR结合的分子动力学模型,并对这个模型中的配体与受体相互作用机制进行了理论研究。
在对模型的结构研究和模拟计算中发现,静电相互作用是配体与受体相互作用的主要作用力。不同配体分子的空间结构和与受体的不同结合方式,会直接影响二聚后两EPO受体分子间夹角和距离,进而产生不同的活性作用——激活剂或者拮抗剂。
本文在对此模型的结构分析和配体与受体结合机制研究的理论基础上,设计了一系列新的EPO小肽类模拟物,我们将其命名为MBPs。使用电荷和疏水性质更强的部分氨基酸来取代原有的EMP中的相应氨基酸。经过计算分析,这些新的模拟肽MBPs在理论上应具有更好的结合和激活活性。
为了从实验上验证设计后的MBPs的结合与聚合活性,我们利用荧光标记法以及微孔板荧光技术建立了EPO模拟物的体外结合与聚合活性检测方法。
由活性检测所得到的实验结果,与理论计算和设计相比较,验证了我们建立的模型的可靠性及计算方法的合理性。为小肽类药物设计和开发开拓了一个新的设计思路和实践方法。
Activation of transmembrane receptors for cytokines occurs when oligomerization of receptor chains is induced by binding of a protein ligand (cytokine) to a specific ligand-binding domain on the receptor. The resulting clustering of tyrosine kinase domains on the cytoplasmic side of the receptor initiates signal transduction events. Receptors may also be activated by bivalent receptor antibodies and by dimeric peptides that interact with the ligand binding domain. Thus activation of receptors by small,peptidyl molecules might make it possible to develop cytokine mimetics that may replace natural counterparts that are now on the market for therapy. This is illustrated here for erythropoietin (EPO). EPO,used for stimulating production of red blood cells,is a major product in biotechnology with world-wide sales of $2.6 billion already in 1995. Thus there has been an increasing interest in molecular mimicry with EPO potency for obviously huge commercial
引文
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[39] Daemen MA, van’t Veer C, Denecker G, et al. J Clin Invest,1999,104 :541-549.
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[41] Chattopadhyay A, Choudhury TD, Bandyopadhyay D, et al. Biochem Phar2macol,2000,59 (4) :419-425.
[42] J uul SE, Ledbetter DJ, Joyco AE, et al. Gut,2001,49 (2) :182-189.
[43] Sasaki R, Masuda S, Nagao M. News PhysiolSci,2001,16 :110-113.
[44] Stuben G, Thews O, Pottgen C, et al. J Cancer Res Clin Oncol,2001,127 (6) :346-350.
[45] Wu SC, Lin SL, Jeng FR.. Scand J Urol Nephrol,2001,35 (2) :136-140.
[46] Dillard DG, Venkatraman G, Cohen C, et al.. Acta Otolaryngol,2001,121 (2) :149-152.
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[49] WrightonNC,Farrell FX,Chang R,etal..Science,1996273:458-464
[50] LivnahO,Stura EA,Johnson DL,etal. Science,1996;273:464-471
[51] JohnsonDL,Farrel lFX,Barbone FP,etal..ChemBiol,1997;4:939-950
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