Structural and biophysical characterization of the 40 kDa PEG-interferon-α2a and its individual positional isomers
Structural and biophysical characterization of the 40 kDa PEG-interferon-α2a and its individual positional isomers
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DOI：
10.1021/bc049781
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发表时间：
2005-05-01
影响因子：
4.7
通讯作者：
Senn, H
中科院分区：
文献类型：
--
作者：
Dhalluin, C;Ross, A;Senn, H
人重组干扰素-α（2a）（IFN α（2a））是治疗各种类型的癌症和病毒性疾病（包括肝炎B/C感染）的有效药物（Roferon-A）。为了改善药物的药理学性质，开发了一种新的聚乙二醇化形式的IFN α（2a）（PEGASYS）。该40 kDa PEG-缀合的IFN α（2a）（（40）PEG-IFN α（2a））通过一个40 kDa支链PEG-聚合物与IFN α（2a）的赖氨酸侧链共价结合而获得。（40）PEG-IFN α（2a）主要是六种分别在K31、K134、K131、K121、K164和K70处修饰的双PEG化位置异构体的混合物。在这里，我们报告了详细的结构和生物物理特性（40）PEG-IFNU α（2a）和它的位置异构体，与IFN α（2a）相比，使用NMR光谱，分析超离心，圆二色性，荧光光谱和差示扫描量热法。我们的结果表明IFN α（2a）的三维结构。在构成（40）PEG-1FN α（2a）的所有位置异构体中，不因聚合物的存在而改变。无论PEG-聚合物附着在何处，它都采用非常移动的和柔性的无规卷曲构象，为蛋白质产生屏蔽而不永久覆盖蛋白质表面。流体动力学数据表明，蛋白质连接的PEG具有比游离PEG-聚合物稍微更紧凑的无规卷曲结构。我们的研究结果还提供了证据的聚乙二醇化所赋予的显着的结构和物理化学的优势：增加的有效流体动力学体积和修改的分子形状，更高的温度稳定性，并减少聚集的趋势。这些结果具有巨大的药理学意义和益处，如PEGASYS的临床所示。这项研究构成了聚乙二醇化蛋白质表征的新标准，并使在分子水平上理解（40）PEG-IFN α（2a）及其位置异构体与其细胞受体的结合迈出了重要一步。
The human recombinant Interferon-alpha(2a) (IFN alpha(2a)) is a potent drug (Roferon-A) to treat various types of cancer and viral diseases including Hepatitis B/C infections. To improve the pharmacological properties of the drug, a new pegylated form of IFN alpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFN alpha(2a) ((40)PEG-IFN alpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFN alpha(2a). (40)PEG-IFN alpha(2a) is a mixture of mainly six monopegylated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively. Here we report the detailed structural and biophysical characterization of (40)PEG-IFNU alpha(2a) and its positional isomers, in comparison with IFN alpha(2a), using NMR spectroscopy, analytical ultracentrifugation, circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry. Our results show that the three-dimensional structure of IFN alpha(2a). is not modified by the presence of the polymer in all positional isomers constituting (40)PEG-lFN alpha(2a). Regardless of where the PEG-polymer is attached, it adopts a very mobile and flexible random coil conformation, producing a shield for the protein without a permanent coverage of the protein surface. Hydrodynamic data indicate that the protein-attached PEG has a slightly more compact random-coil structure than the free PEG-polymer. Our results also provide evidence of significant structural and physicochemical advantages conferred by the pegylation: increase of the effective hydrodynamic volume and modification of the molecular shape, higher temperature stability, and reduced tendency for aggregation. These results are of tremendous pharmacological interest and benefit as was clinically shown with PEGASYS. This study constitutes a new standard for the characterization of pegylated proteins and enables an important step toward the understanding on a molecular level of the binding of (40)PEG-IFN alpha(2a), and its positional isomers to its cellular receptors.