Protein Engineering Unit, Life and Science Research Department, Anti-Doping Lab-Qatar (ADLQ), Doha, Qatar; Drug Design Group, Department of Pharmacy, University of Groningen, Groningen, Netherlands.
Cairo University, Faculty of Science, Chemistry Department, Giza, Egypt.
Biomed Pharmacother. 2019 Apr;112:108725. doi: 10.1016/j.biopha.2019.108725. Epub 2019 Feb 28.
Glucarpidase, also known as carboxypeptidase G, is a Food and Drug Administration-approved enzyme used in targeted cancer strategies such as antibody-directed enzyme prodrug therapy (ADEPT). It is also used in drug detoxification when cancer patients have excessive levels of the anti-cancer agent methotrexate. The application of glucarpidase is limited by its potential immunogenicity and limited catalytic efficiency. To overcome these pitfalls, mutagenesis was applied to the glucarpidase gene of Pseudomonas sp. strain RS-16 to isolate three novels "biobetter" variants with higher specific enzyme activity. DNA sequence analysis of the genes for the variants showed that each had a single point mutation, resulting in the amino acid substitutions: I100 T, G123S and T239 A. K V and K measurements confirmed that each variant had increased catalytic efficiency relative to wild type glucarpidase. Additionally, circular dichroism studies indicated that they had a higher alpha-helical content relative to the wild type enzyme. However, three different software packages predicted that they had reduced protein stability, which is consistent with having higher activities as a tradeoff. The novel glucarpidase variants presented in this work could pave the way for more efficient drug detoxification and might allow dose escalation during chemotherapy. They also have the potential to increase the efficiency of ADEPT and to reduce the number of treatment cycles, thereby reducing the risk that patients will develop antibodies to glucarpidase.
葡糖醛酸酶，又称羧肽酶 G，是一种获得美国食品和药物管理局批准的酶，用于抗体导向酶前药治疗（ADEPT）等靶向癌症策略。当癌症患者甲氨蝶呤水平过高时，它也用于药物解毒。葡糖醛酸酶的应用受到其潜在免疫原性和有限的催化效率的限制。为了克服这些缺陷，对假单胞菌 RS-16 株的葡糖醛酸酶基因进行了诱变，分离出三种新型“生物改良”变体，具有更高的比酶活。变体基因的 DNA 序列分析表明，每种变体都有一个单点突变，导致氨基酸取代：I100T、G123S 和 T239A。K V 和 K 测量证实，与野生型葡糖醛酸酶相比，每种变体都具有更高的催化效率。此外，圆二色性研究表明，与野生型酶相比，它们具有更高的α-螺旋含量。然而，三个不同的软件包预测它们的蛋白质稳定性降低，这与更高的活性是一种权衡。本文介绍的新型葡糖醛酸酶变体为更有效的药物解毒铺平了道路，并可能允许在化疗期间增加剂量。它们还有潜力提高 ADEPT 的效率，并减少治疗周期，从而降低患者对葡糖醛酸酶产生抗体的风险。
