Increasing methotrexate resistance by combination of active-site mutations in human dihydrofolate reductase
Increasing methotrexate resistance by combination of active-site mutations in human dihydrofolate reductase
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DOI：
10.1016/j.jmb.2007.07.076
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发表时间：
2007-10-26
影响因子：
5.6
通讯作者：
Pelletier, Joelle N.
中科院分区：
文献类型：
--
作者：
Volpato, Jordan P.;Fossati, Elena;Pelletier, Joelle N.
甲氨蝶呤抗性形式的人二氢叶酸还原酶有可能保护健康细胞免受甲氨蝶呤 (MTX) 的毒性，从而改善癌症治疗期间的预后。已经表明，通过组合两个独立地赋予弱MTX抗性的活性位点突变可以获得协同MTX抗性。为了获得用于该应用的更多高度抗MTX的人二氢叶酸还原酶（hDHFR）变体，我们使用半理性方法来获得对MTX高度抗性的hDHFR组合活性位点突变体。我们创建了一个编码残基 Phe31、Phe34 和 GIn35 处的各种氨基酸的组合突变体文库。体内文库选择是在含有高浓度 MTX 的培养基上的细菌系统中实现的。我们对残基 31、34 和 35 处具有不同氨基酸组合的 10 个新型 MTX 抗性突变体进行了表征。纯化突变体的动力学和抑制参数表明，较高的 MTX 抗性大致与更多的突变相关，最高抗性突变体包含三个活性位点突变 (K-i(MTX) = 59-180 nM；野生型 K-i(MTX) < 0.03 nM）。在电阻和催化效率之间观察到负相关，其下降主要是由于对底物二氢叶酸的 Km 增加。我们通过将最具耐药性的突变体转染到 DHFR 敲除的 CHO 细胞中，验证了 MTX 耐药性 hDHFR 可以保护真核细胞免受 MTX 毒性。转染的变体在 MTX 浓度比野生型酶高 100 倍至 4000 倍以上时能够存活，这是最具抵抗力的三重突变体，提供的保护超出了培养基中可包含的 MTX 的最大浓度。 hDHFR 的这些高度耐药变体在癌症治疗中使用 MTX 期间提供了骨髓保护的潜力。 (c) 2007 Elsevier Ltd. 保留所有权利。
Methotrexate-resistant forms of human dihydrofolate reductase have the potential to protect healthy cells from the toxicity of methotrexate (MTX), to improve prognosis during cancer therapy. It has been shown that synergistic MTX-resistance can be obtained by combining two active-site mutations that independently confer weak MTX-resistance. In order to obtain more highly MTX-resistant human dihydrofolate reductase (hDHFR) variants for this application, we used a semi-rational approach to obtain combinatorial active-site mutants of hDHFR that are highly resistant towards MTX. We created a combinatorial mutant library encoding various amino acids at residues Phe31, Phe34 and GIn35. In vivo library selection was achieved in a bacterial system on medium containing high concentrations of MTX. We characterized ten novel MTX-resistant mutants with different amino acid combinations at residues 31, 34 and 35. Kinetic and inhibition parameters of the purified mutants revealed that higher MTX-resistance roughly correlated with a greater number of mutations, the most highly-resistant mutants containing three active site mutations (K-i(MTX) = 59-180 nM; wild-type K-i(MTX) < 0.03 nM). An inverse correlation was observed between resistance and catalytic efficiency, which decreased mostly as a result of increased Km toward the substrate dihydrofolate. We verified that the MTX-resistant hDHFRs can protect eukaryotic cells from MTX toxicity by transfecting the most resistant mutants into DHFR-knockout CHO cells. The transfected variants conferred survival at concentrations of MTX between 100-fold and >4000-fold higher than the wild-type enzyme, the most resistant triple mutant offering protection beyond the maximal concentration of MTX that could be included in the medium. These highly resistant variants of hDHFR offer potential for myeloprotection during administration of MTX in cancer treatment. (c) 2007 Elsevier Ltd. All rights reserved.