Department of Medical Biotechnology, School of Advanced Medical Science, Tabriz University of Medical Sciences,Tabriz, Iran.
Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
Iran J Med Sci. 2021 Nov;46(6):454-467. doi: 10.30476/ijms.2020.86984.1705.
Ocriplasmin has been developed for the induction of posterior vitreous detachment in patients with vitreomacular adhesion. At physiological pH, ocriplasmin is susceptible to autolytic and proteolytic degradation, limiting its activity duration. These undesirable properties of ocriplasmin can be reduced by site-directed mutagenesis, so that its enzymatic activities can be augmented. This study aimed to design ocriplasmin variants with improved biological/physicochemical characteristics via bioinformatics tools.
This study was performed in Tabriz University of Medical Sciences, Tabriz, Iran, 2019. Through site-directed mutagenesis, three ocriplasmin variants were designed. Structural analysis was performed on the wild-type variant and the mutant variants using the Protein Interactions Calculator (PIC) server. The interactions between the S-2403 substrate and the ocriplasmin variants were studied by molecular docking simulations, and binding capability was evaluated by the calculation of free binding energy. The conformational features of protein-substrate complex systems for all the variants were evaluated using molecular dynamic simulations at 100 nanoseconds.
The structural analysis of ocriplasmin revealed that the substitution of threonine for alanine 59 significantly reduced proteolytic activity, while the substitution of glutamic acid for lysine 156 influenced autolytic function. The molecular docking simulation results indicated the appropriate binding of the substrate to the ocriplasmin variants with high-to-low affinities. The binding affinity of the wild-type variant for the substrate was higher than that between the mutant variants and the substrate. Simulation analyses, consisting of the root-mean-square deviation, the root-mean-square fluctuation, and the center-of-mass average distance showed a higher affinity of the substrate for the wild type than for the mutant variants.
The mutational analysis of ocriplasmin revealed that A59T and K156E mutagenesis could be used for the development of a new variant with higher therapeutic efficacy.
Ocriplasmin 已被开发用于诱导玻璃体黄斑粘连患者的后玻璃体脱离。在生理 pH 值下，Ocriplasmin 易发生自溶和蛋白水解降解，从而限制了其活性持续时间。通过定点突变可以降低 Ocriplasmin 的这些不理想性质，从而增强其酶活性。本研究旨在通过生物信息学工具设计具有改善的生物学/物理化学特性的 Ocriplasmin 变体。
本研究于 2019 年在伊朗大不里士医科大学进行。通过定点突变，设计了三种 Ocriplasmin 变体。使用蛋白质相互作用计算器（PIC）服务器对野生型变体和突变变体进行结构分析。通过分子对接模拟研究 S-2403 底物与 Ocriplasmin 变体之间的相互作用，并通过计算自由结合能评估结合能力。使用分子动力学模拟在 100 纳秒内评估所有变体的蛋白质-底物复合体系的构象特征。
Ocriplasmin 的结构分析表明，苏氨酸取代丙氨酸 59 显著降低了蛋白水解活性，而谷氨酸取代赖氨酸 156 影响了自溶功能。分子对接模拟结果表明，底物与 Ocriplasmin 变体的结合合适，亲和力从高到低。野生型变体与底物的结合亲和力高于突变变体与底物的结合亲和力。包括均方根偏差、均方根波动和质心平均距离在内的模拟分析表明，底物与野生型的亲和力高于与突变变体的亲和力。
Ocriplasmin 的突变分析表明，A59T 和 K156E 突变可用于开发具有更高治疗效果的新型变体。
