Allosteric lipoxygenase effectors as potential drugs

花生四烯酸脂氧合酶（ALOX）及其代谢产物（类花生酸和相关物质）不仅参与细胞成熟和分化，而且参与炎症、过度增殖和神经系统疾病的发病机制。在过去的二十年里，科学家们已经开发出了阻止类花生酸生物合成或抑制这些介质与相应受体相互作用的药物。如今，抗炎性COX 2抑制剂（COXIB）是全球最畅销的药物之一，并且已经开发了ALOX 5途径的抑制剂以及白三烯受体（cysLTR 1）拮抗剂。其中一些药物（齐留通、孟鲁司特、扎鲁司特）目前可作为抗哮喘药物处方。人类基因组计划的完成表明存在六种功能性ALOX基因（ALOX 5、ALOX 15、ALOX 15 B、ALOX 12、ALOX 12 B、ALOX 3），其编码六种不同的ALOX同工酶。对于其中的五种，包括ALOX 15和ALOX 5敲除小鼠，这些动物的实验表明不同ALOX同种型的不同生物学功能。对于ALOX 15，已经报道了促炎和抗炎特性（ALOX 15的双重作用），并且最近详细的动力学研究表明人ALOX 15表现出变构特性。这一发现是相当令人惊讶的，因为哺乳动物ALOX 15直系同源物的晶体结构表明，酶由单一多肽链组成，并且没有证据表明存在保守的变构配体结合位点。在水溶液中，ALOX 15形成由催化单体和调节单体组成的催化活性二聚体，所述催化单体在活性位点结合底物脂肪酸，所述调节单体充当变构配体的结合位点。ALOX 15反应的变构特性为开发异构体特异性ALOX 15效应物（抑制剂和激活剂）提供了可能性，这是目前不可用的。当我们探索几种取代的吲哚基苯基氨基磺基氨基甲酸酯的生物活性时，我们发现它们选择性地阻止ALOX 15依赖性亚油酸氧化。相反，花生四烯酸的氧化没有被抑制。详细的动力学测量，分析的反应产物和初步的结构建模的基础上的ALOX 15的X-射线坐标表明，这些底物特异性效应结合在底物结合口袋的变构单体。这种结合诱导第二催化单体的构象变化，其抑制亚油酸但不抑制花生四烯酸的氧化。这些数据共同表明了开发底物特异性变构ALOX 15效应物的可能性。考虑到ALOX 15在炎症中的双重功能，寻找变构ALOX 15效应物（抑制剂和激活剂）可能比常规抑制剂筛选策略更有前途。

Arachidonic acid lipoxygenases (ALOXs) and their metabolites (eicosanoids and related substances) have been implicated in cell maturation and differentiation, but also in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Over the past twenty years scientists have developed drugs preventing the biosynthesis of eicosanoids or inhibiting the interaction of these mediators with the corresponding receptors. Today anti-inflammatory COX2 inhibitors (COXIBs) are among the most selling drugs worldwide and inhibitors of the ALOX5 pathway as well as leukotriene receptor (cysLTR1) antagonists have been developed. Some of them (Zileuton, Montelukast, Zafirlukast) are currently available for prescription as anti-asthmatic drugs. Completion of the human genome project indicated the existence of six functional ALOX genes (ALOX5, ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3), which encode for six different ALOX-isozymes. For five of them including ALOX15 and ALOX5 knockout mice are available and experiments with these animals indicated distinct biological functions for the different ALOX isoforms. For ALOX15 pro- and anti-inflammatory properties (dual role of ALOX15) have been reported and detailed kinetic investigations recently suggested that human ALOX15 exhibits allosteric properties. This finding was rather surprising since the crystal structures of mammalian ALOX15 orthologs have indicated that the enzymes consist of a single polypeptide chain and there was no evidence for a conserved allosteric ligand binding site. In aqueous solutions ALOX15 forms catalytically active dimers consisting of a catalytic monomer, which binds the substrate fatty acid at the active site, and a regulatory monomer serving as binding site for allosteric ligands. The allosteric character of the ALOX15 reaction offers the possibility for the development of isoform-specific ALOX15 effectors (inhibitors and activators), which are currently not available.When we explored the biological activities of several substituted indolylphenylaminosulfocarbamates we found them to selectively prevent ALOX15 dependent linoleic acid oxygenation. In contrast, oxidation of arachidonic acid was not inhibited. Detailed kinetic measurements, analysis of the reaction products and preliminary structural modelling on the basis of the ALOX15 X-ray coordinates suggested that these substrate specific effectors bind at the substrate-binding pocket of the allosteric monomer. This binding induces a conformational change of the second catalytic monomer, which inhibits oxygenation of linoleic acid but not of arachidonic acid. Together these data indicate the possibility for the development of substrate-specific allosteric ALOX15 effectors. Considering the dual functionality of ALOX15 in inflammation the search for allosteric ALOX15 effectors (inhibitors and activators) may be more promising than conventional inhibitor screening strategies.