Dhalluin Christophe, Ross Alfred, Huber Walter, Gerber Paul, Brugger Doris, Gsell Bernard, Senn Hans
F. Hoffmann-La Roche AG, Department of Pharma Research, CH-4070 Basel, Switzerland.
Bioconjug Chem. 2005 May-Jun;16(3):518-27. doi: 10.1021/bc049780h.
Type-I Interferons exert antiviral and antiproliferative activities through the binding to a common cell surface receptor comprising two subunits, IFNAR1 and IFNAR2. Human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) used to treat various cancers and viral diseases including Hepatitis B/C infections. To significantly improve the pharmacological properties of the drug, a pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side-chain of IFNalpha(2a). Here, we report the detailed structural, kinetic, and thermodynamic analysis of the binding to the extracellular domain of the receptor IFNAR2 of (40)PEG-IFNalpha(2a) and its isolated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively, in comparison with unmodified IFNalpha(2a). Our binding studies, using the surface plasmon resonance technique, show that the pegylation does not abolish the binding to the receptor, but significantly reduces the affinity mainly due to a change of the association rate. The results are supported by modeling and simulation of the binding, using Self-Avoiding-Walk calculations for the polymer conformations. A correlation between the structural parameters and the kinetic and thermodynamic parameters of the binding of the positional isomers could be established. For the Isomer-K31 and -K164, the PEG-polymer attachment point is located in proximity to the binding interface, and the isomers display affinity in the range 150-520 nM in an enthalpy-driven binding process. In contrast for the Isomer-K134, -K131, -K121, and -K70, the PEG-polymer is attached remotely from the binding interface, and the isomers exhibit a higher affinity (32-76 nM) in an entropy-driven binding process. This study constitutes an essential collection of knowledge on which the interaction of (40)PEG-IFNalpha(2a) and its positional isomers with its cellular receptors can be better understood.
I型干扰素通过与由IFNAR1和IFNAR2两个亚基组成的共同细胞表面受体结合来发挥抗病毒和抗增殖活性。人重组干扰素α(2a)（IFNα(2a)）是一种用于治疗包括乙型/丙型肝炎感染在内的各种癌症和病毒性疾病的有效药物（罗扰素-A）。为了显著改善该药物的药理学特性，开发了一种聚乙二醇化形式的IFNα(2a)（派罗欣）。这种40 kDa的聚乙二醇共轭IFNα(2a)（(40)PEG-IFNα(2a)）是通过将一个40 kDa的支链聚乙二醇聚合物共价结合到IFNα(2a)的赖氨酸侧链上获得的。在此，我们报告了(40)PEG-IFNα(2a)及其分别在K31、K134、K131、K121、K164和K70处修饰的分离位置异构体与受体IFNAR2的细胞外结构域结合的详细结构、动力学和热力学分析，并与未修饰的IFNα(2a)进行比较。我们使用表面等离子体共振技术进行的结合研究表明，聚乙二醇化不会消除与受体的结合，但主要由于缔合速率的变化而显著降低了亲和力。聚合物构象的自回避行走计算对结合进行建模和模拟，支持了这些结果。可以建立位置异构体结合的结构参数与动力学和热力学参数之间的相关性。对于异构体-K31和-K164，聚乙二醇聚合物连接点位于靠近结合界面的位置，并且异构体在焓驱动的结合过程中显示出150 - 520 nM范围内的亲和力。相比之下，对于异构体-K134、-K131、-K121和-K70，聚乙二醇聚合物远离结合界面连接，并且异构体在熵驱动的结合过程中表现出更高的亲和力（32 - 76 nM）。这项研究构成了关于(40)PEG-IFNα(2a)及其位置异构体与其细胞受体相互作用的重要知识集合，有助于更好地理解它们之间的相互作用。
