De novo engineering of a human cystathionine-ýý-lyase for systemic (L)-Methionine depletion cancer therapy.
De novo engineering of a human cystathionine-ýý-lyase for systemic (L)-Methionine depletion cancer therapy.
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用于全身（L）-甲硫氨酸耗竭癌症治疗的人胱硫醚-α-裂解酶的从头工程。
DOI：
10.1021/cb300335j
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发表时间：
2012
影响因子：
4
通讯作者：
Georgiou,George
中科院分区：
文献类型：
--
作者：
Stone,Everett;Paley,Olga;Hu,Jian;Ekerdt,Barbara;Cheung,Nai-Kong;Georgiou,George
近半个世纪以来，人们就知道人类肿瘤，包括源自神经系统的肿瘤，如胶质母细胞瘤、髓母细胞瘤和神经母细胞瘤，比正常组织对蛋氨酸 (l-Met) 饥饿更敏感。最近，通过给予恶臭假单胞菌甲硫氨酸-γ-裂解酶（MGL）进行全身性Met消耗可以有效抑制小鼠异种移植的人类肿瘤。然而，细菌衍生的 MGL 在血清中不稳定 (t1/2= 1.9 ± 0.2 h)，并且在灵长类动物中具有高度免疫原性。由于人类基因组不编码人类 MGL 酶，因此我们通过重新设计结构同源的磷酸吡哆醛依赖性人类酶胱硫醚-γ-裂解酶 (hCGL)，从头创建了蛋氨酸降解酶。 hCGL 降解 1-胱硫醚，但对 1-Met 没有表现出混杂活性。合理的设计和扫描饱和诱变导致产生了含有三个氨基酸取代的变体（hCGL-NLV），该变体用 akcat/KM of 5.6 × 102M–1s–1 降解 l-Met，并表现出血清失活t1/2= 78 ± 5 h（非聚乙二醇化）。在体外，hCGL-NLV 对 14 种神经母细胞瘤细胞系的细胞毒性基本上是与P的没有区别。恶臭MGL。小鼠静脉注射聚乙二醇化 hCGL-NLV 在 30 多小时内将血清 Met 从 123 μM 降低至 <5 μM。重要的是，用聚乙二醇化 hCGL-NLV 治疗神经母细胞瘤小鼠异种移植物导致肿瘤生长几乎完全停止。由于 hCGL-NLV 的作用方式不需要突破血脑屏障，因此该酶可能对中枢神经系统产生或转移的敏感肿瘤有潜在的应用。
It has been known for nearly a half century that human tumors, including those derived from the nervous system such as glioblastomas, medulloblastoma, and neuroblastomas are much more sensitive than normal tissues tol-methionine (l-Met) starvation. More recently, systemicl-Met depletion by administration ofPseudomonas putidamethionine-γ-lyase (MGL) could effectively inhibit human tumors xenografted in mice. However, bacterial-derived MGLs are unstable in serum (t1/2= 1.9 ± 0.2 h) and highly immunogenic in primates. Since the human genome does not encode a human MGL enzyme, we createdde novoa methionine degrading enzyme by reengineering the structurally homologous pyridoxal phosphate-dependent human enzyme cystathionine-γ-lyase (hCGL). hCGL degradesl-cystathionine but displays no promiscuous activity towardl-Met. Rational design and scanning saturation mutagenesis led to the generation of a variant containing three amino acid substitutions (hCGL-NLV) that degradedl-Met with akcat/KMof 5.6 × 102M–1s–1and displayed a serum deactivationt1/2= 78 ± 5 h (non-PEGylated).In vitro, the cytotoxicity of hCGL-NLV toward 14 neuroblastoma cell lines was essentially indistinguishable from that of theP. putidaMGL. Intravenous administration of PEGylated hCGL-NLV in mice reduced seruml-Met from 123 μM to <5 μM for over 30 h. Importantly, treatment of neuroblastoma mouse xenografts with PEGylated hCGL-NLV resulted in near complete cessation of tumor growth. Since the mode of action of hCGL-NLV does not require breaching the blood-brain barrier, this enzyme may have potential application for sensitive tumors that arise from or metastasize to the central nervous system.