Crystal Structures of Three Classes of Non-Steroidal Anti-Inflammatory Drugs in Complex with Aldo-Keto Reductase 1C3

Aldo-keto reductase 1C3 (AKR1C3) catalyses the NADPH dependent reduction of carbonyl groups in a number of important steroid and prostanoid molecules. The enzyme is also over-expressed in prostate and breast cancer and its expression is correlated with the aggressiveness of the disease. The steroid products of AKR1C3 catalysis are important in proliferative signalling of hormone-responsive cells, while the prostanoid products promote prostaglandin-dependent proliferative pathways. In these ways, AKR1C3 contributes to tumour development and maintenance, and suggest that inhibition of AKR1C3 activity is an attractive target for the development of new anti-cancer therapies. Non-steroidal anti-inflammatory drugs (NSAIDs) are one well-known class of compounds that inhibits AKR1C3, yet crystal structures have only been determined for this enzyme with flufenamic acid, indomethacin, and closely related analogues bound. While the flufenamic acid and indomethacin structures have been used to design novel inhibitors, they provide only limited coverage of the NSAIDs that inhibit AKR1C3 and that may be used for the development of new AKR1C3 targeted drugs. To understand how other NSAIDs bind to AKR1C3, we have determined ten crystal structures of AKR1C3 complexes that cover three different classes of NSAID, N-phenylanthranilic acids (meclofenamic acid, mefenamic acid), arylpropionic acids (flurbiprofen, ibuprofen, naproxen), and indomethacin analogues (indomethacin, sulindac, zomepirac). The N-phenylanthranilic and arylpropionic acids bind to common sites including the enzyme catalytic centre and a constitutive active site pocket, with the arylpropionic acids probing the constitutive pocket more effectively. By contrast, indomethacin and the indomethacin analogues sulindac and zomepirac, display three distinctly different binding modes that explain their relative inhibition of the AKR1C family members. This new data from ten crystal structures greatly broadens the base of structures available for future structure-guided drug discovery efforts.This work was funded by Lottery Health Research (CJS; grant number 265027), the Auckland Medical Research Foundation (JUF and CJS; grant number 1110004. JUF; grant number 1109008), the National eScience Infrastructure (JUF), and the Maurice Wilkins Centre for Molecular Biodiscovery Flexible Research Seeding Programme (JUF and CJS). We further acknowledge salary support from the Maurice Wilkins Centre for Molecular Biodiscovery (CJS, JUF) and Summer Studentship funding from the Faculty of Science, University of Auckland (CJS, RMT).

Beamline: MX2
EPN: 3432z
PDB: 3R94
Organism: Homo sapiens
Expression System: Escherichia coli
Sequence: >3R94:A|PDBID|CHAIN|SEQUENCE MDSKHQCVKLNDGHFMPVLGFGTYAPPEVPRSKALEVTKLAIEAGFRHIDSAHLYNNEEQVGLAIRSKIADGSVKREDIF YTSKLWSTFHRPELVRPALENSLKKAQLDYVDLYLIHSPMSLKPGEELSPTDENGKVIFDIVDLCTTWEAMEKCKDAGLA KSIGVSNFNRRQLEMILNKPGLKYKPVCNQVECHPYFNRSKLLDFCKSKDIVLVAYSALGSQRD

醛酮还原酶1C3（Aldo-keto reductase 1C3, AKR1C3）可催化依赖于烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate, NADPH）的多种重要甾体及类前列腺素分子中羰基的还原反应。该酶在前列腺癌与乳腺癌组织中存在过表达现象，且其表达水平与疾病的侵袭性呈正相关。醛酮还原酶1C3催化生成的甾体产物在激素响应细胞的增殖信号通路中发挥重要作用，而其类前列腺素产物则可促进依赖于前列腺素的增殖通路。基于上述机制，该酶可促进肿瘤的发生与维持，这提示抑制醛酮还原酶1C3的活性是开发新型抗癌疗法的极具潜力的靶点。
非甾体抗炎药（Non-steroidal anti-inflammatory drugs, NSAIDs）是一类已知的可抑制醛酮还原酶1C3的化合物，但目前仅解析出该酶与氟芬那酸、吲哚美辛及其紧密相关类似物结合的晶体结构。尽管基于氟芬那酸与吲哚美辛的晶体结构已被用于设计新型抑制剂，但这类结构对可抑制醛酮还原酶1C3、且可用于开发醛酮还原酶1C3靶向新药的非甾体抗炎药的覆盖范围仍十分有限。
为阐明其他非甾体抗炎药与醛酮还原酶1C3的结合机制，本研究解析了10组醛酮还原酶1C3复合物的晶体结构，覆盖了三类不同的非甾体抗炎药：N-苯基邻氨基苯甲酸类（甲氯芬那酸、甲芬那酸）、芳基丙酸类（氟比洛芬、布洛芬、萘普生）以及吲哚美辛类似物类（吲哚美辛、舒林酸、佐美酸）。N-苯基邻氨基苯甲酸类与芳基丙酸类化合物可结合至酶的共同位点，包括酶催化中心与组成型活性位点口袋，其中芳基丙酸类化合物能更有效地探查组成型口袋。与之形成对比的是，吲哚美辛及其类似物舒林酸与佐美酸则呈现出三种截然不同的结合模式，这解释了它们对醛酮还原酶1C家族成员的相对抑制活性差异。
本次解析的10组晶体结构数据极大地拓展了可用于后续结构导向药物开发的结构库。
本研究得到了以下基金的资助：彩票健康研究项目（CJS；项目编号265027）、奥克兰医学研究基金会（JUF与CJS；项目编号1110004；JUF；项目编号1109008）、国家电子科学基础设施项目（JUF）以及莫里斯·威尔金斯分子生物发现中心灵活研究种子基金项目（JUF与CJS）。本研究同时感谢莫里斯·威尔金斯分子生物发现中心为CJS与JUF提供的薪酬支持，以及奥克兰大学理学院为CJS与RMT提供的暑期学生资助金。
光束线：MX2
EPN编号：3432z
蛋白质数据库（Protein Data Bank, PDB）编号：3R94
来源物种：智人（Homo sapiens）
表达系统：大肠杆菌（Escherichia coli）
序列：>3R94:A|PDBID|CHAIN|SEQUENCE MDSKHQCVKLNDGHFMPVLGFGTYAPPEVPRSKALEVTKLAIEAGFRHIDSAHLYNNEEQVGLAIRSKIADGSVKREDIF YTSKLWSTFHRPELVRPALENSLKKAQLDYVDLYLIHSPMSLKPGEELSPTDENGKVIFDIVDLCTTWEAMEKCKDAGLA KSIGVSNFNRRQLEMILNKPGLKYKPVCNQVECHPYFNRSKLLDFCKSKDIVLVAYSALGSQRD