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[摘要]：It 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 to L-methionine (L-Met)starvation. More recently, systemic L-Met depletion by administration of Pseudomonas putida methionine-gamma-lyase (MGL) could effectively inhibit human tumors xenografted in mice. However, bacterial-derived MGLs are unstable in serum (t(1/2) = 1.9 +/- 0.2 h) and highly immunogenic in primates. Since the human genome does not encode a human MGL enzyme, we created de novo a methionine degrading enzyme by : reengineering the structurally homologous pyridoxal phosphate-dependent human enzyme cystathionine-gamma-lyase (hCGL). hCGL degrades L-cystathionine but displays no promiscuous activity toward L-Met. Rational design and scanning saturation mutagenesis led to the generation of a variant containing three amino acid substitutions (hCGL-NLV) that degraded L-Met with a k(cat)/K-M of 5.6 x 10(2) M-1 s(-1) and displayed a serum deactivation t(1/2) = 78 +/- 5 h (non-PEGylated). In vitro, the cytotoxicity of hCGL-NLV toward 14 neuroblastoma cell lines was essentially indistinguishable from that of the P. putida MGL. Intravenous administration of PEGylated hCGL-NLV in mice reduced serum L-Met from 123 mu M to <5 mu M 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. |
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