Iqbal Ambar, Abbas Wasim, Ejaz Samina, Riaz Naheed, Ashok Avinash Karkada, Hayat Muhammad Munawar, Ashraf Muhammad
Institute of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan; Department of Biochemistry, Institute of Biochemistry, Biotechnology & Bioinformatics (IBBB), B.J. Campus, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan.
Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, Punjab, Pakistan.
Int J Biol Macromol. 2025 Jun;314:143665. doi: 10.1016/j.ijbiomac.2025.143665. Epub 2025 Apr 30.
Lipoxygenase (LOX) and cyclooxygenase (COX) pathways generate biologically active mediators implicated in inflammatory disorders and several classes of cancer. Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the COX pathway by inhibiting the COX-1 and COX-2 enzymes. We reported earlier that several NSAIDs, including naproxen, aspirin and acetaminophen, inhibited lipoxygenase (LOX) enzyme at sub-micromolar concentrations. In continuation, the present work demonstrates the anti-LOX activity of nine more NSAIDs supported by in vitro, in silico, MD simulation and breast cancer cell line studies. All tested drugs displayed potent to excellent inhibitory profiles with IC values <24.93 ± 0.64 μM. Aceclofenac (IC 0.85 ± 0.06 μM) was the most active drug, followed by indomethacin (IC 1.13 ± 0.07 μM), meloxicam (IC 1.94 ± 0.07 μM) and ketorolac (IC 9.26 ± 0.82 μM). Celecoxib (IC 15.81 ± 0.71 μM), lornoxicam (IC 16.54 ± 0.28 μM) and nimesulide (IC 19.87 ± 0.85 μM) showed excellent inhibitory profiles. Flurbiprofen (IC 21.73 ± 0.93 μM) and etoricoxib (IC 24.93 ± 0.64 μM) moderately inhibited the target enzyme. SAR studies revealed that active molecules decorated with the carboxylate group afforded strong binding interactions as observed by in vitro assays and structural features. Other drugs, including enol derivatives and celecoxib, also showcased enhanced binding interactions. However, the cytotoxic effects of NSAIDs against the MCF-7 breast cancer cell line did not disclose significant anticancer activity. Molecular docking studies against human 5-LOX offered the best binding affinities for aceclofenac (-13.54 kcal/mol), accompanied by conventional hydrogen bonding and hydrophobic interactions as supported by the in vitro results. Docking studies with DNA dodecamer established minor groove binding with their possible role in DNA replication and gene expression. Density functional theory (DFT) and ESP studies, MD simulations and MMPBSA free energy calculations further reiterated the stability of ligand-receptor complexes. Overall, these findings highlight the potential of targeted NSAIDs as dual COX/LOX inhibitors with broader therapeutic relevance in inflammatory disorders.
脂氧合酶（LOX）和环氧化酶（COX）途径可产生与炎症性疾病和几类癌症相关的生物活性介质。非甾体抗炎药（NSAIDs）通过抑制COX-1和COX-2酶来抑制COX途径。我们之前报道过，包括萘普生、阿司匹林和对乙酰氨基酚在内的几种NSAIDs在亚微摩尔浓度下可抑制脂氧合酶（LOX）。在此基础上，本研究通过体外、计算机模拟、分子动力学（MD）模拟和乳腺癌细胞系研究，证明了另外九种NSAIDs的抗LOX活性。所有测试药物均表现出强效至优异的抑制活性，IC值<24.93±0.64μM。醋氯芬酸（IC 0.85±0.06μM）是活性最强的药物，其次是吲哚美辛（IC 1.13±0.07μM）、美洛昔康（IC 1.94±0.07μM）和酮咯酸（IC 9.26±0.82μM）。塞来昔布（IC 15.81±0.71μM）、氯诺昔康（IC 16.54±0.28μM）和尼美舒利（IC 19.87±0.85μM）表现出优异的抑制活性。氟比洛芬（IC 21.73±0.93μM）和依托考昔（IC 24.93±0.64μM）对目标酶有中度抑制作用。构效关系（SAR）研究表明，如体外实验和结构特征所示，带有羧基的活性分子能产生强烈的结合相互作用。其他药物，包括烯醇衍生物和塞来昔布，也表现出增强的结合相互作用。然而，NSAIDs对MCF-7乳腺癌细胞系的细胞毒性作用并未显示出显著的抗癌活性。针对人5-LOX的分子对接研究表明，醋氯芬酸具有最佳的结合亲和力（-13.54 kcal/mol），体外实验结果支持其存在传统氢键和疏水相互作用。与DNA十二聚体的对接研究确定了它们在小沟结合中的作用及其在DNA复制和基因表达中的可能作用。密度泛函理论（DFT）和静电势（ESP）研究、MD模拟以及分子力学泊松-玻尔兹曼表面面积（MMPBSA）自由能计算进一步证实了配体-受体复合物的稳定性。总体而言，这些发现突出了靶向NSAIDs作为双COX/LOX抑制剂在炎症性疾病中具有更广泛治疗意义的潜力。
