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首页> 外文期刊>The FEBS journal >Insights into the slow-onset tight-binding inhibition of Escherichia coli dihydrofolate reductase: detailed mechanistic characterization of pyrrolo [3,2-f] quinazoline-1,3-diamine and its derivatives as novel tight-binding inhibitors
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Insights into the slow-onset tight-binding inhibition of Escherichia coli dihydrofolate reductase: detailed mechanistic characterization of pyrrolo [3,2-f] quinazoline-1,3-diamine and its derivatives as novel tight-binding inhibitors

机译：对大肠杆菌二氢叶酸还原酶缓慢发作的紧密结合抑制的见解：吡咯并[3,2-f]喹唑啉-1,3-二胺及其衍生物作为新型紧密结合抑制剂的详细机理表征

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Dihydrofolate reductase (DHFR) is a pivotal enzyme involved in the de novo pathway of purine synthesis, and hence, represents an attractive target to disrupt systems that require rapid DNA turnover. The enzyme acquires resistance to available drugs by various molecular mechanisms, which necessitates the continuous discovery of novel antifolates. Previously, we identified a set of novel molecules that showed binding to E. coli DHFR by means of a thermal shift without establishing whether they inhibited the enzyme. Here, we show that a fraction of those molecules represent potent and novel inhibitors of DHFR activity. 7-[(4-aminophenyl) methyl]-7H-pyrrolo [3,2-f] quinazoline-1,3-diamine, a molecule with no reported inhibition of DHFR, potently inhibits the enzyme with a K-i value of 7.42 +/- 0.92 nM by competitive displacement of the substrate dihydrofolic acid. It shows uncompetitive inhibition vis-a-vis NADPH, indicating that the inhibitor has markedly increased affinity for the NADPH-bound form of the enzyme. Further, we demonstrate that the mode of binding of the inhibitor to the enzyme-NADPH binary complex conforms to the slow-onset, tight-binding model. By contrast, mechanistic characterization of the parent molecule 7H-pyrrolo [3,2-f] quinazoline-1,3-diamine shows that lack of (4-aminophenyl)-methyl group at the seventh position abolishes the slow onset of inhibition. This finding provides novel insights into the role of substitutions on inhibitors of E. coli DHFR and represents the first detailed kinetic investigation of a novel diaminopyrrolo-quinazoline derivative on a prokaryotic DHFR. Furthermore, marked differences in the potency of inhibition for E. coli and human DHFR makes this molecule a promising candidate for development as an antibiotic.
机译：二氢叶酸还原酶（DHFR）是参与嘌呤从头合成途径的关键酶，因此，它代表了一个有吸引力的靶标，可破坏需要快速DNA转换的系统。该酶通过各种分子机制获得对可用药物的抗性，这需要不断发现新的抗叶酸药物。以前，我们鉴定了一组新型分子，这些分子通过热转移显示出与大肠杆菌DHFR的结合，而没有确定它们是否抑制酶。在这里，我们表明这些分子的一小部分代表了DHFR活性的有效抑制剂。 7-[（（4-氨基苯基）甲基] -7H-吡咯并[3,2-f]喹唑啉-1,3-二胺是一种没有报道抑制DHFR的分子，其Ki值为7.42 + /有效抑制了该酶。 -通过竞争置换底物二氢叶酸获得0.92 nM。它相对于NADPH表现出非竞争性的抑制作用，表明该抑制剂对NADPH结合形式的酶的亲和力显着提高。此外，我们证明抑制剂与酶-NADPH二元复合物的结合模式符合慢速发作，紧密结合模型。相比之下，母体分子7H-吡咯并[3,2-f]喹唑啉-1,3-二胺的机械表征显示，在第七位缺少（4-氨基苯基）-甲基消除了缓慢的抑制作用。该发现提供了对大肠杆菌DHFR抑制剂中取代作用的新颖见解，并代表了对原核DHFR上的新型二氨基吡咯并喹唑啉衍生物的首次详细动力学研究。此外，对大肠杆菌和人DHFR的抑制能力的显着差异使该分子成为发展为抗生素的有希望的候选者。

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