Ho Kwok Ki, Mukhopadhyay Abhijit, Li Yi Feng, Mukhopadhyay Soma, Weiner Henry
Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, IN 47907-2063, United States.
Biochem Pharmacol. 2008 Sep 1;76(5):690-6. doi: 10.1016/j.bcp.2008.06.018. Epub 2008 Jul 4.
Cyclophosphamides are pro-drugs whose killing agent is produced from an aldehyde that is formed by the action of a P450 oxidation step. The mustard from the aldehyde can destroy bone marrow cells as well as the tumor. Aldehyde dehydrogenase (EC 1.2.1.3) can oxidize the aldehyde and hence inactivate the cytotoxic intermediate but bone marrow has little, if any, of the enzyme. Others have shown that over-expression of the enzyme can afford protection of the marrow. A T186S mutant of the human stomach enzyme (ALDH3) that we developed has increased activity against the aldehyde compared to the native enzyme and HeLa cells transformed with the point mutant are better protected against the killing effect of the drug. It took threefold more drug to kill 90% of the cells transformed with the mutant compared to the native enzyme (15.8 compared to 5.1mM of a precursor of the toxic aldehyde). Analysis of molecular models makes it appear that removing the methyl group of threonine in the T186S mutant allows the bulky aldehyde to bind better. The mutant was found to be a poorer enzyme when small substrates such as benzaldehyde derivatives were investigated. Thus, the enzyme appears to be better only with large substrates such as the one produced by cyclophosphamide.
环磷酰胺是前体药物，其杀伤剂由一种醛生成，该醛通过P450氧化步骤产生。醛衍生出的芥子气既能破坏骨髓细胞，也能破坏肿瘤细胞。醛脱氢酶（EC 1.2.1.3）可氧化该醛，从而使细胞毒性中间体失活，但骨髓中几乎没有这种酶（即便有也极少）。其他人已表明，该酶的过表达可保护骨髓。我们研发的人胃酶（ALDH3）的T186S突变体与天然酶相比，对醛的活性有所增强，用该点突变体转化的HeLa细胞对药物的杀伤作用具有更好的抗性。与天然酶相比，杀死90%用突变体转化的细胞所需的药物剂量是其三倍（有毒醛前体的浓度分别为15.8mM和5.1mM）。分子模型分析表明，T186S突变体中苏氨酸的甲基去除后，可使体积较大的醛更好地结合。当研究苯甲醛衍生物等小分子底物时，发现该突变体是一种较差的酶。因此，该酶似乎仅对环磷酰胺产生的这类大分子底物表现出更好的活性。
