Engineered Charge Redistribution of Gp2 Proteins through Guided Diversity for Improved PET Imaging of Epidermal Growth Factor Receptor.
Engineered Charge Redistribution of Gp2 Proteins through Guided Diversity for Improved PET Imaging of Epidermal Growth Factor Receptor.
复制标题
通过引导多样性设计 Gp2 蛋白的电荷重新分布，以改善表皮生长因子受体的 PET 成像。
DOI：
10.1021/acs.bioconjchem.8b00144
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发表时间：
2018
影响因子：
4.7
通讯作者：
Hackel,BenjaminJ
中科院分区：
文献类型：
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作者：
Case,BrettA;Kruziki,MaxA;Johnson,SadieM;Hackel,BenjaminJ
Gp2 结构域是用于合成配体工程的蛋白质支架。然而，天然蛋白质功能导致保守表面上电荷的异质分布，这可能阻碍进一步的开发和利用。我们的目标是在不减弱功能的情况下调节电荷，这对于小蛋白质来说是一个挑战，因为每个突变都是蛋白质结构的重要组成部分。我们构建了具有电荷中和或电荷翻转突变的合理引导的组合文库，并通过酵母展示和流式细胞术对它们进行分类，以确保稳定性和靶标结合。功能变体的深度测序揭示了克隆依赖性环境中以及广泛的表皮生长因子受体 (EGFR)、胰岛素受体和免疫球蛋白 G 结合物中的有效突变。功能突变体每个结构域平均有 4.3 个电荷中和突变，同时保持净负电荷。我们开发了一种靶向 EGFR 的 Gp2 突变体，其电荷密度降低了 33%，保持了净电荷，并改善了电荷分布均匀性，同时提高了热稳定性 (Tm= 87 ± 1 °C)、提高了结合特异性并保持了亲和力 (Kd= 8.8 ± 0.6 nM)。该分子与 1,4,7-三氮杂环壬烷,1-戊二酸-4,7-乙酸缀合进行 64 Cu 螯合，并评估异种移植 A431 (EGFRhigh) 和 MDA-MB-435 (EGFRlow) 肿瘤小鼠中的生理分布。切除的组织伽马计数和正电子发射断层扫描/计算机断层扫描成像显示注射后 2 小时具有良好的 EGFRhightumor 信号 (4.7 ± 0.5%ID/g)，分子特异性通过 EGFRlowtumors 的低摄取证明 (0.6 ± 0.1%ID/g，显着低于非电荷修饰的 Gp2，p= 0.01)。这些结果为改进的 Gp2 框架提供了电荷突变，验证了电荷工程的有效方法，并提高了分子成像的生理 EGFR 靶向性能。
The Gp2 domain is a protein scaffold for synthetic ligand engineering. However, the native protein function results in a heterogeneous distribution of charge on the conserved surface, which may hinder further development and utility. We aim to modulate charge, without diminishing function, which is challenging in small proteins where each mutation is a significant fraction of protein structure. We constructed rationally guided combinatorial libraries with charge-neutralizing or charge-flipping mutations and sorted them, via yeast display and flow cytometry, for stability and target binding. Deep sequencing of functional variants revealed effective mutations both in clone-dependent contexts and broadly across binders to epidermal growth factor receptor (EGFR), insulin receptor, and immunoglobulin G. Functional mutants averaged 4.3 charge neutralizing mutations per domain while maintaining net negative charge. We evolved an EGFR-targeted Gp2 mutant that reduced charge density by 33%, maintained net charge, and improved charge distribution homogeneity while elevating thermal stability (Tm= 87 ± 1 °C), improving binding specificity, and maintaining affinity (Kd= 8.8 ± 0.6 nM). This molecule was conjugated with 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid for64Cu chelation and evaluated for physiological distribution in mice with xenografted A431 (EGFRhigh) and MDA-MB-435 (EGFRlow) tumors. Excised tissue gamma counting and positron emission tomography/computed tomography imaging revealed good EGFRhightumor signal (4.7 ± 0.5%ID/g) at 2 h post-injection and molecular specificity evidenced by low uptake in EGFRlowtumors (0.6 ± 0.1%ID/g, significantly lower than for non-charge-modified Gp2,p= 0.01). These results provide charge mutations for an improved Gp2 framework, validate an effective approach to charge engineering, and advance performance of physiological EGFR targeting for molecular imaging.