Inhibition of MIF bioactivity by rational design of pharmacological inhibitors of MIF tautomerase activity
Inhibition of MIF bioactivity by rational design of pharmacological inhibitors of MIF tautomerase activity
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DOI：
10.1021/jm010534q
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发表时间：
2002-06-06
影响因子：
7.3
通讯作者：
Al-Abed, Y
中科院分区：
文献类型：
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作者：
Dios, A;Mitchell, RA;Al-Abed, Y
促炎症介质巨噬细胞移动抑制因子(MIF)由免疫和内分泌细胞产生，抑制糖皮质激素的抗炎活性。MIF还催化非自然存在的多巴色素、苯丙酮酸和某些儿茶酚胺的D-异构体的互变异构化，这表明MIF可能通过底物上的酶作用发挥其生物学效应。然而，目前还没有发现与MIF相关的生理底物。定点突变研究并不一致地支持将完整的、功能的催化位点作为MIF生物活性的先决条件。我们假设，催化活性部位，而不是酶活性本身，在MIF促炎活性中起着关键作用。因此，我们设计了小的类药物分子，结合在MIF的催化活性互变消除酶位置，并测试了MIF的生物活性。在这里，我们描述了一类通过将氨基酸偶联到一系列苯甲醛衍生物上而产生的亚胺结合物的合理设计和合成，这些苯甲醛衍生物可以抑制MIF互换消失酶和生物活性。我们发现芳香族氨基酸Schiff碱比脂肪族氨基酸Schiff碱更能抑制MIF的酶活性和生物活性。例如，芳香族氨基酸Schiff碱甲酯对MIF互变构象酶活性的IC50抑制作用在1.65-50um之间，这表明芳香族残基在活性部位附近的额外结合起着关键作用。2-[(4-羟基亚甲基)氨基]3-(1H-吲哚-3-基)丙酸甲酯(8)对MIF互变异构酶活性的抑制作用最强，其IC50为1.65um。我们发现，化合物8与MIF活性部位结合后，在三种已建立的生物检测中均能抑制MIF的生物活性：ERK-1/2 MAP激酶激活、P53依赖的细胞凋亡和血清饥饿细胞的增殖。通过流式细胞仪检测，化合物8抑制了MIF与其尚未鉴定的同源细胞表面受体的相互作用，表明互变构酶活性部位在受体结合中起着关键作用。因此，化合物8对MIF生物活性的抑制作用与对MIF互换错构酶活性的抑制密切相关，这一联系以前没有通过使用小分子MIF抑制剂来实现。氨基酸-苯甲醛Schiff碱型MIF拮抗剂的抑制活性是对MIF细胞生物活性抑制剂进行有意义的结构/功能分析的第一步。
The pro-inflammatory mediator macrophage migration inhibitory factor (MIF) is produced by immune and endocrine cells and inhibits the antiinflammatory activities of glucocorticoids. MIF also catalyzes the tautomerization of the non-naturally occurring D-isomer of dopachrome, phenylpyruvate, and certain catecholamines, suggesting that MIF might exert its biological effects via enzymatic action on a substrate. However, no physiologically relevant substrate for MIF has been identified. Site-directed mutagenesis studies have not consistently supported a requirement for an intact, functional catalytic site as a prerequisite for MIF bioactivity. We hypothesized that the catalytically active site, but not the enzymatic activity per se, nevertheless plays a critical role in MIF pro-inflammatory activity. Accordingly, we designed small druglike molecules that bind at the catalytically active tautomerase site of MIF and tested the complex for MIF bioactivity. We describe herein the rational design and synthesis of a class of imine conjugates produced by coupling amino acids to a range of benzaldehyde derivatives that inhibit MIF tautomerase and biological activities. We found that aromatic amino acid Schiff bases were better inhibitors of MIF enzymatic and bioactivities compared to the aliphatic ones. For instance, the IC50 inhibition of MIF tautomerase activity by aromatic amino acid Schiff base methyl esters was achieved at a concentration between 1.65 and 50 muM, suggesting a critical role for the additional binding of the aromatic residues within the vicinity of the active site. The most potent inhibitor of MIF tautomerase activity was 2-[(4-hydroxybenzylidene)amino] 3-(1H-indol-3-yl)propionic acid methyl ester (8), with an IC50 of 1.65 muM. We found that compound 8 binding to MIF active site resulted in the inhibition of MIF bioactivity in three established bioassays: ERK-1/2 MAP kinase activation, p53-dependent apoptosis, and proliferation of serum-starved cells. Compound 8 inhibited MIF interaction with its as yet unidentified cognate cell surface receptor as shown by flow cytometry, concluding a critical role for the tautomerase active site in receptor binding. Thus the inhibitory effect of compound 8 on MIF bioactivities strongly correlated with the inhibition of MIF tautomerase activity, a connection not made previously through use of small-molecule MIF inhibitors. The inhibitory activity of amino acid-benzaldehyde Schiff base-type MIF antagonists is the first step toward a meaningful structure/function analysis of inhibitors of MIF cellular bioactivities.