Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enzyme active form
Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enzyme active form
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DOI：
10.1021/jm030843b
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发表时间：
2003-09-11
影响因子：
7.3
通讯作者：
Neidle, S
中科院分区：
文献类型：
--
作者：
Johansson, E;Parkinson, GN;Neidle, S
大肠杆菌硝基还原酶 (NTR) 已广泛用于自杀基因治疗（GDEPT 和 ADEPT）应用，作为二硝基苯甲酰胺类硝基芳香族前药的激活酶。 NTR 先前已被证明是具有两个活性位点的同型二聚体酶。我们在此展示了 NTR 还原形式及其与抑制剂双香豆素和三种二硝基苯甲酰胺前药的复合物的晶体结构。天然酶的氧化形式和还原形式的结构比较表明，主要的结构变化发生在FMN辅因子中，并表明酶本身是相对刚性的结构，主要提供发生氢化物转移的刚性结构框架。氮丙啶基二硝基苯甲酰胺前药 CB 1954 在同二聚酶的两个活性位点上以不同的方式结合，同时采用疏水性和（在活性位点 B 中）与 Lys14 侧链的直接氢键接触。在活性位点 A 中，2-硝基基团堆积在 FMN 上方，而在活性位点 B 中，4-硝基基团堆积在 FMN 上方，这解释了为什么观察到任一硝基基团的还原。相比之下，二硝基苯甲酰胺芥子化合物 SN 23862 的较大芥子基团迫使前药在两个活性位点结合，只有 2-硝基能够参与从 FMN 的氢化物转移，这解释了为什么只观察到 2-羟胺还原产物。在每个位点，前药的硝基与酶直接形成氢键接触；在活性位点A中，2-硝基与Ser40接触，4-硝基与Asn71接触，而在活性位点B中，2-硝基与Thr41的主链氮接触，4-硝基与Lys14侧链接触。相关的酰胺取代芥子 SN 27217 以大致相似的方式结合，但较大的酰胺基取代基能够到达并接触 Arg107 的侧链，进一步限制了结合位点的前药构象。抑制剂双香豆素似乎主要通过π堆积相互作用和疏水接触结合，酶中没有构象变化。其中一个羟基香豆素亚基通过与异咯嗪环π重叠而堆叠在FMN平面上方，深入到凹槽中，而另一个则不太明确。这些研究为进一步的前药设计提供了指导。环上的空间体积（例如，芥子而不是氮丙啶）可以限制可能的结合方向，并且可还原的硝基必须位于芥子的对位。甲酰胺侧链上的取代仍然允许前药结合，但也限制了它们在结合位点的方向。最后，通过改变酶的结构而不是前药的结构来调节底物特异性可能有效地集中于修饰 Phe124 残基及其周围的残基。
The E. coli nitroreductase enzyme (NTR) has been widely used in suicide gene therapy (GDEPT and ADEPT) applications as a activating enzyme for nitroaromatic prodrugs of the dinitrobenzamide class. NTR has been previously shown to be a homodimeric enzyme with two active sites. We present here the crystal structures of the reduced form of NTR and its complexes with the inhibitor dicoumarol and three dinitrobenzamide prodrugs. Comparison of the structures of the oxidized and reduced forms of the native enzyme shows that the principal structural changes occur in the FMN cofactor and indicate that the enzyme itself is a relatively rigid structure that primarily provides a rigid structural framework on which hydride transfer occurs. The aziridinyldinitrobenzamide prodrug CB 1954 binds in nonidentical ways in both of the two active sites of the homodimeric enzyme, employing both hydrophobic and (in active site B) a direct H-bond contact to the side chain of Lys14. In active site A the 2-nitro group stacks above the FMN, and in active site B the 4-nitro group does, explaining why reduction of either nitro group is observed. In contrast, the larger mustard group of the dinitrobenzamide mustard compound SN 23862 forces the prodrug to bind at both active sites with only the 2-nitro group able to participate in hydride transfer from the FMN, explaining why only the 2-hydroxylamine reduction product is observed. In each site, the nitro groups of the prodrug make direct H-bond contacts with the enzyme; in active Site A the 2-nitro to Ser40 and the 4-nitro to Asn71, while in active Site B the 2-nitro contacts the main chain nitrogen of Thr41 and the 4-nitro group the Lys14 side chain. The related amide-substituted mustard SN 27217 binds in a broadly similar fashion, but with the larger amide group substituent able to reach and contact the side chain of Arg107, further restricting the prodrug conformations in the binding site. The inhibitor dicoumarol appears to bind primarily by pi-stacking interactions and hydrophobic contacts, with no conformational changes in the enzyme. One of the hydroxycoumarin subunits stacks above the plane of the FMN via pi-overlap with the isoalloxazine ring, penetrating deep into the groove, with the other less well-defined. These studies suggest guidelines for further prodrug design. Steric bulk (e.g., mustard rather than aziridine) on the ring can limit the possible binding orientations, and the reducible nitro group must be located para to the mustard. Substitution on the carboxamide side chain still allows the prodrugs to bind, but also limits their orientation in the binding site. Finally, modulating substrate specificity by alteration of the structure of the enzyme rather than the prodrug might usefully focus on modifying the Phe124 residue and those surrounding it.