Department of Pharmaceutical Sciences & Center for Pharmaceutical Biotechnology, Skaggs School of Pharmacy and Pharmaceutical Sciences , University of Colorado Anschutz Medical Campus , Aurora , Colorado 80045 , United States.
Mol Pharm. 2019 Nov 4;16(11):4621-4635. doi: 10.1021/acs.molpharmaceut.9b00719. Epub 2019 Oct 2.
Two of the most common forms of chemical modifications that compromise the efficacy of therapeutic proteins are the deamidation of asparagine residues and oxidation of methionine residues. We probed how deamidation affects the structure, stability, aggregation, and function of interferon alpha-2a (IFNA2a), and compared with our earlier results on methionine oxidation. Upon deamidation, no significant changes were observed in the global secondary structure of IFNA2a with minor changes in its tertiary structure. However, deamidation destabilized the protein, and increased its propensity to aggregate under accelerated stress conditions. Cytopathic inhibition and antiproliferation assays showed drastic decrease in the functionality of deamidated IFNA2a compared to the wild-type. 2D NMR measurements showed structural changes in local protein regions, with no effect on the overall global structure of IFNA2a. These local protein regions corresponded well with the aggregation hot-spots predicted by computational programs, and the functional hot-spots identified by site-directed mutagenesis. When compared to the effects of methionine oxidation, deamidation caused lesser aggregation, because of lesser structural unfolding observed in aggregation hot-spots by 2D NMR. In comparison to oxidation, deamidation showed larger decrease in function, because deamidation affected key amino acid residues in functional hot-spots as observed by 2D NMR and structural modeling. Such quantitative comparison between the effects of deamidation and oxidation on a pharmaceutical protein has not been done before, and the high-resolution structural information on local protein regions obtained by 2D NMR provided a better insight compared to low-resolution methods that probe global protein structure.
两种最常见的影响治疗性蛋白质疗效的化学修饰形式是天冬酰胺残基的脱酰胺和蛋氨酸残基的氧化。我们探讨了脱酰胺如何影响干扰素α-2a（IFNA2a）的结构、稳定性、聚集和功能，并与我们之前关于蛋氨酸氧化的结果进行了比较。脱酰胺后，IFNA2a 的整体二级结构没有明显变化，三级结构略有变化。然而，脱酰胺使蛋白质不稳定，并在加速应激条件下增加其聚集的倾向。细胞病变抑制和增殖测定显示，与野生型相比，脱酰胺的 IFNA2a 功能明显下降。2D NMR 测量显示局部蛋白质区域的结构变化，对 IFNA2a 的整体全局结构没有影响。这些局部蛋白质区域与计算程序预测的聚集热点以及定点突变鉴定的功能热点非常吻合。与蛋氨酸氧化的影响相比，脱酰胺引起的聚集较少，因为 2D NMR 观察到聚集热点的结构展开较少。与氧化相比，脱酰胺的功能下降更大，因为 2D NMR 和结构建模观察到脱酰胺影响功能热点中的关键氨基酸残基。以前没有对药物蛋白的脱酰胺和氧化的影响进行过这种定量比较，2D NMR 获得的局部蛋白质区域的高分辨率结构信息比探测整体蛋白质结构的低分辨率方法提供了更好的见解。
