羧肽酶 G2 循环排列工程,用于设计一种自动抑制酶。

Brahm J Yachnin, Sagar D Khare
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引用次数: 0

摘要

羧肽酶 G2(CPG2)是美国食品和药物管理局(FDA)批准的一种酶制剂药物,用于治疗接受 MTX 治疗的癌症患者的甲氨蝶呤(MTX)毒性。它还被用于定向酶促化疗,但这种策略因循环酶对原药的异位激活而受到阻碍。开发一种可被肿瘤蛋白酶激活的 CPG2 将有助于这些应用。我们报告了一种基于蛋白酶可及性的筛选方法的开发情况,该方法可识别出靠近 CPG2 活性位点的循环置换候选位点。由此产生的六种环状变体在表达、结构、热稳定性以及四种情况下的活性水平上与野生型酶相似。我们根据使用 Rosetta 软件获得的 permutants 结构模型合理地解释了这些结果。我们开发了一种基于细胞生长的选择系统,并证明了当与外质定向信号肽融合时,我们的一种环状 permutants 能以与野生型酶相同的效率在大肠杆菌中产生 MTX 抗性。由于包涵体具有与野生型 CPG2 相似的特性,这些酶是开发用于治疗的自抑制、蛋白酶可激活的酶原形式 CPG2 的良好起点。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Engineering carboxypeptidase G2 circular permutations for the design of an autoinhibited enzyme.

Carboxypeptidase G2 (CPG2) is an Food and Drug Administration (FDA)-approved enzyme drug used to treat methotrexate (MTX) toxicity in cancer patients receiving MTX treatment. It has also been used in directed enzyme-prodrug chemotherapy, but this strategy has been hampered by off-site activation of the prodrug by the circulating enzyme. The development of a tumor protease activatable CPG2, which could be achieved using a circular permutation of CPG2 fused to an inactivating 'prodomain', would aid in these applications. We report the development of a protease accessibility-based screen to identify candidate sites for circular permutation in proximity of the CPG2 active site. The resulting six circular permutants showed similar expression, structure, thermal stability, and, in four cases, activity levels compared to the wild-type enzyme. We rationalize these results based on structural models of the permutants obtained using the Rosetta software. We developed a cell growth-based selection system, and demonstrated that when fused to periplasm-directing signal peptides, one of our circular permutants confers MTX resistance in Escherichia coli with equal efficiency as the wild-type enzyme. As the permutants have similar properties to wild-type CPG2, these enzymes are promising starting points for the development of autoinhibited, protease-activatable zymogen forms of CPG2 for use in therapeutic contexts.

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