Department of Chemistry-BMC, Uppsala University, Uppsala, Sweden.
Department of Chemistry-BMC, Uppsala University, Uppsala, Sweden; Department of Neurochemistry, Stockholm University, Stockholm, Sweden.
Adv Cancer Res. 2014;122:199-244. doi: 10.1016/B978-0-12-420117-0.00006-2.
The prodrug azathioprine is primarily used for maintaining remission in inflammatory bowel disease, but approximately 30% of the patients suffer adverse side effects. The prodrug is activated by glutathione conjugation and release of 6-mercaptopurine, a reaction most efficiently catalyzed by glutathione transferase (GST) A2-2. Among five genotypes of GST A2-2, the variant A2E has threefold-fourfold higher catalytic efficiency with azathioprine, suggesting that the expression of A2E could boost 6-mercaptopurine release and adverse side effects in treated patients. Structure-activity studies of the GST A2-2 variants and homologous alpha class GSTs were made to delineate the determinants of high catalytic efficiency compared to other alpha class GSTs. Engineered chimeras identified GST peptide segments of importance, and replacing the corresponding regions in low-activity GSTs by these short segments produced chimeras with higher azathioprine activity. By contrast, H-site mutagenesis led to decreased azathioprine activity when active-site positions 208 and 213 in these favored segments were mutagenized. Alternative substitutions indicated that hydrophobic residues were favored. A pertinent question is whether variant A2*E represents the highest azathioprine activity achievable within the GST structural framework. This issue was addressed by mutagenesis of H-site residues assumed to interact with the substrate based on molecular modeling. The mutants with notably enhanced activities had small or polar residues in the mutated positions. The most active mutant L107G/L108D/F222H displayed a 70-fold enhanced catalytic efficiency with azathioprine. The determination of its structure by X-ray crystallography showed an expanded H-site, suggesting improved accommodation of the transition state for catalysis.
前药巯嘌呤主要用于维持炎症性肠病的缓解，但约 30%的患者出现不良反应。该前药通过谷胱甘肽结合和释放 6-巯基嘌呤而被激活，该反应最有效地由谷胱甘肽转移酶 (GST) A2-2 催化。在 GST A2-2 的五个基因型中，变体 A2E 对巯嘌呤的催化效率提高了三倍至四倍，这表明 A2E 的表达可以增加治疗患者中 6-巯基嘌呤的释放和不良反应。对 GST A2-2 变体和同源α类 GST 的结构-活性研究阐明了与其他α类 GST 相比具有高催化效率的决定因素。工程化嵌合体确定了 GST 肽段的重要性，并用这些短片段替换低活性 GST 中的相应区域，产生了具有更高巯嘌呤活性的嵌合体。相比之下，当这些有利片段中的活性位点位置 208 和 213 发生突变时，H 位点突变导致巯嘌呤活性降低。替代取代表明，疏水性残基是有利的。一个相关的问题是变体 A2*E 是否代表 GST 结构框架内可实现的最高巯嘌呤活性。通过突变假定基于分子建模与底物相互作用的 H 位点残基来解决这个问题。具有显著增强活性的突变体在突变位置具有小或极性残基。活性最高的突变体 L107G/L108D/F222H 对巯嘌呤的催化效率提高了 70 倍。其结构的 X 射线晶体学测定显示 H 位点扩大，表明对催化过渡态的容纳能力得到改善。
