Fluorescent inhibitors for IspF, an enzyme in the non-mevalonate pathway for isoprenoid biosynthesis and a potential target for antimalarial therapy
Fluorescent inhibitors for IspF, an enzyme in the non-mevalonate pathway for isoprenoid biosynthesis and a potential target for antimalarial therapy
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DOI：
10.1002/anie.200503003
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Diederich, F
中科院分区：
文献类型：
--
作者：
Crane, CM;Kaiser, J;Diederich, F
安徽1069号。化学。内部艾德2006，45,1069-1074 2006 Wiley-VCH Verlag GmbH&Co.KGaA，Weinheim各种疟原虫的多重耐药菌株，迫切需要寻找具有新作用模式的新疗法。[1]非甲戊酸途径中的酶，用于将C5前体组装成萜烯，异戊烯基二磷酸(IPP)1和二甲基烯丙基二磷酸(DMAPP)2，最近被确定为抗疟疾和抗微生物药物的靶标。3]非甲氧丙戊酸途径[4]的特征是丙酮酸3和甘油醛3-磷酸4的缩合(方案1)，并且是高等植物[5a-c]和许多细菌[4b，5c，6]中1和2的唯一来源，包括一些引起严重疾病的细菌，如结核分枝杆菌[6b]和原生动物疟原虫寄生虫(Apicomplexa)。[3]由于哺乳动物只利用甲氧戊酸途径，[6]开发抑制非甲戊酸途径酶的小分子先导化合物可能是迈向新的抗疟疾药物的关键步骤。3]我们选择了ISPF(2C-甲基-D-赤藓糖醇2，4-环二磷酸合成酶，YGBB)作为基于结构的铅生成的靶标，[7，8]ISPF是非甲戊酸途径中的第五个酶，它催化4-二磷酸-2C-甲基-D-赤藓糖醇2-磷酸(5)环化成关键的环状二磷酸中间体2C-甲基-D-赤藓糖醇2，4-环二磷酸(6)。已发表的晶体结构(蛋白质数据库(PDB)[8，9]代码1GX1和1JY8)表明ISPF是C3对称的同源三聚体。拓扑上等价的活性中心位于相邻亚基的界面上。一个单体的刚性、保守的“PocketIII”与5的胞苷部分结合，而相邻单体的更大、更灵活的“Pocket II”与5和6的2C-甲基-d-赤藓糖醇部分结合(关于这些口袋中的蛋白质残基，见图1b)。后一个口袋还包含一个四面体配位的锌II离子。占据两个口袋的抑制剂7-9是在分子建模软件MOLOC的帮助下设计的。[10，11]在没有已知抑制剂的情况下，我们在第一步选择保持天然底物的CDP部分以占据口袋III，同时通过适当大小的连接物将二磷酸连接到芳香族残基，以占据由Leu 76‘，Phe 61’和Ile 57‘定义的PocketII的疏水裂隙(图1)。由于在第一阶段，我们也有兴趣开发一种基于荧光的酶抑制试验，荧光邻氨基苯甲酸酯(2-氨基苯甲酸酯)和丹磺酰(5，5-二甲氨基苯磺酰基)残基被选为到达Pocket II的芳香族部分。
1069 Angew. Chem. Int. Ed. 2006, 45, 1069–1074 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim multiple-drug-resistant strains of the various Plasmodium parasites, the search for new therapies with novel modes of action is of urgent necessity.[1] Enzymes in the non-mevalonate pathway, utilized for assembling the C5 precursors to terpenes, isopentenyl diphosphate (IPP) 1 and dimethylallyl diphosphate (DMAPP) 2, were recently identified as targets for antimalarial and antimicrobial drugs.[2, 3] The non-mevalonate pathway [4] is characterized by the condensation of pyruvate 3 and glyceraldehyde 3-phosphate 4 (Scheme 1) and is the sole source for 1 and 2 in plastids of higher plants [5a–c] and in many bacteria [4b, 5c, 6] including some responsible for serious diseases such as Mycobacterium tuberculosis [6b] and the protozoan Plasmodium parasites (Apicomplexa).[3] Since mammals exclusively utilize the mevalonate pathway,[6] the development of small-molecule lead compounds inhibiting the enzymes of the non-mevalonate pathway may be a key step towards new antimalarial drugs.[1b, 2, 3] We have chosen the enzyme IspF (2C-methyl-d-erythritol 2, 4-cyclodiphosphate synthase, ygbB) as a target for structure-based lead generation.[7, 8] IspF is the fifth enzyme in the non-mevalonate pathway and catalyzes the cyclization of 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate (5), to the key cyclic diphosphate intermediate, 2C-methyl-d-erythritol 2, 4-cyclodiphosphate (6). Published crystal structures (Protein Data Bank (PDB)[8, 9] codes 1GX1 and 1JY8) show IspF to be a C3-symmetric homotrimer. The topologically equivalent active sites are located at the interfaces of adjacent subunits. The rigid, well-conserved “PocketIII” of one monomer binds the cytidine moiety of 5, and the larger, more flexible “Pocket II” of an adjacent monomer binds the 2C-methyl-d-erythritol moieties of 5 and 6 (for the protein residues lining these pockets, see Figure1b). The latter pocket also contains a tetrahedrally coordinated ZnII ion.Inhibitors 7–9, occupying both pockets, were designed with the help of the molecular modeling software MOLOC.[10, 11] In the absence of known inhibitors, we chose in a first step to maintain the CDP moiety of the natural substrate to occupy Pocket III while connecting the diphosphate by means of an appropriately sized linker to an aromatic residue, for occupation of the hydrophobic cleft of PocketII, defined by Leu 76’, Phe 61’, and Ile 57’(Figure 1). Since at a first stage, we were also interested in developing a fluorescence-based enzyme inhibition assay, fluorescent anthranilate (2-aminobenzoate) and dansyl (5, 5-dimethylaminonaphthalenesulfamoyl) residues were chosen as aromatic moieties reaching into Pocket II.[12]