Structural analysis of a holoenzyme complex of mouse dihydrofolate reductase with NADPH and a ternary complex with the potent and selective inhibitor 2,4-diamino-6-(2′-hydroxydibenz[b,f]-azepin-5-yl)methylpteridine
Structural analysis of a holoenzyme complex of mouse dihydrofolate reductase with NADPH and a ternary complex with the potent and selective inhibitor 2,4-diamino-6-(2′-hydroxydibenz[b,f]-azepin-5-yl)methylpteridine
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DOI：
10.1107/s0907444908022348
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发表时间：
2008-09-01
影响因子：
2.2
通讯作者：
Rosowsky, Andre
中科院分区：
文献类型：
--
作者：
Cody, Vivian;Pace, Jim;Rosowsky, Andre
研究表明，芳基上含有O-羧基的2，4-二氨基-6-芳基甲基蝶啶和2，4-二氨基-5-芳基嘧啶类化合物对二氢叶酸还原酶(DHFR)有较强的选择性抑制作用，这些二氢叶酸还原酶来自于引起HIV/AIDS患者肺炎的卡氏肺孢子虫等条件致病菌。为了了解一系列取代二苯并[b，f]氮杂环己烷对叶酸盐的构效关系，测定了小鼠DHFR(mDHFR；哺乳动物同系物)与NADPH和抑制剂2，4-二氨基-6-(2‘-羟基二苯并[b，f]氮杂-5-基)甲基蝶啶的全息和三元配合物的晶体结构，其分辨率分别为1.9和1.4埃。O-(3-羧丙基)抑制剂PT684与三元络合物的结构数据表明，O-羧基烷氧基侧链没有电子密度。侧链要么断裂，要么完全无序。电子密度符合较弱的羟基化合物PT684a。此外，mDHFR与NADPH和效力较低的2‘-(4-羧基苯基)抑制剂PT682的共结晶没有显示出抑制剂的电子密度，导致了首次报道的全酶复合体，尽管多次尝试结晶三元复合体。PT682在mDHFR和P.carinii DHFR(PcDHFR)活性部位的模拟数据表明，结合需要配体诱导的酶构象变化才能使抑制剂适合活性部位，或者抑制剂侧链必须采用与其他羧烷氧基抑制剂不同的结合方式。这些数据还表明，mDHFR三元配合物的活性中心体积减少，反映在螺旋C(残基59-)的相对位移比pcDHFR三元配合物的位移0.6埃。这些数据与对pcDHFR更强的抑制效力一致。
It has been shown that 2,4-diamino-6-arylmethylpteridines and 2,4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2'-hydroxydibenz[b,f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 angstrom resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed no electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2'-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59-64) by 0.6 angstrom compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.