Background: Non-steroidal anti-inflammatory drugs, cyclooxygenase (COX)-2 selective inhibitors, have been explored for prevention and treatment of several inflammatory chronic conditions including arthritis, and cancer. However, the long-term use of these drugs is associated with gastrointestinal, renal, and cardiovascular side effects. Later, COX/5-lipoxygenase (5-LOX) dual inhibitors (eg, licofelone) have been developed but did not enter into the market from the clinical trails due to COX-1/2 inhibition-associated side effects. Hence, targeting microsomal prostaglandin E synthase-1 (mPGES-1) and 5-LOX can be an ideal approach while sparing COX-1/2 activities for development of the next generation of anti-inflammatory drugs with better efficacy and safety.
Materials and Methods: In silico (molecular modelling) studies were used to design a mPGES-1/5-LOX dual inhibitory and COX-1/2 sparing lead molecule licofelone analogue-9 (LFA-9) by modifying the pharmacophore of licofelone. In vitro cell-free enzymatic (mPGES-1, 5-LOX, COX-1/2) assays using fluorometric/colorimetric methods and cell-based assays (LPS-induced PGE₂, LTB₄, and PGI₂ productions from macrophages) using ELISA technique, isothermal calorimetry, and circular dichroism techniques were performed to determine the mPGES-1/5-LOX inhibitory efficacy and selectivity. Anti-inflammatory efficacy of LFA-9 was evaluated using a carrageenan (inflammogen)-induced rat paw edema model. Infiltration/expression of CD68 immune cells and TNF-α in paw tissues were evaluated using confocal microscope and immunoblot analysis. Anti-cancer effect of LFA-9 was evaluated using colon spheroids in vitro.
Results: LFA‐9 inhibited mPGES-1/5-LOX and their products PGE₂ and LTB₄, spared COX-1/2 and its product PGI₂. LFA‐9 bound strongly with human mPGES‐1/5‐LOX enzymes and induced changes in their secondary structure, thereby inhibited their enzymatic activities. LFA-9 inhibited carrageenan-induced inflammation (70.4%) in rats and suppressed CD68 immune cell infiltration (P ≤ 0.0001) and TNF-α expression. LFA-9 suppressed colon tumor stemness (60.2%) in vitro through inhibition of PGE₂ (82%) levels.
Conclusion: Overall study results suggest that LFA-9 is a mPGES-1/5-LOX dual inhibitor and showed anti-inflammatory and colorectal cancer preventive activities, and warranted detailed studies.



中文翻译：

---

一种新型 mPGES-1/5-LOX 双抑制剂 LFA-9 的发现和开发，用于预防和治疗慢性炎症性疾病

背景：非甾体抗炎药、环氧合酶 (COX)-2 选择性抑制剂已被探索用于预防和治疗包括关节炎和癌症在内的多种炎症性慢性疾病。然而，这些药物的长期使用与胃肠道、肾脏和心血管的副作用有关。后来，COX/5-脂氧合酶（5-LOX）双重抑制剂（如licofelone）已经开发出来，但由于COX-1/2抑制相关的副作用，并未从临床试验中进入市场。因此，靶向微粒体前列腺素 E 合酶-1 (mPGES-1) 和 5-LOX 可能是一种理想的方法，同时保留 COX-1/2 的活性，以开发具有更好疗效和安全性的下一代抗炎药。
材料和方法：计算机模拟（分子建模）研究用于通过修饰 licofelone 的药效团来设计 mPGES-1/5-LOX 双重抑制和 COX-1/2 保留先导分子 licofelone 类似物 9 (LFA-9)。_{使用荧光/比色法和基于细胞的测定法（LPS 诱导的 PGE 2}、LTB ₄和 PGI ₂）的体外无细胞酶（mPGES-1、5-LOX、COX-1/2）测定使用ELISA技术、等温量热法和圆二色性技术进行巨噬细胞产生）以确定mPGES-1/5-LOX抑制功效和选择性。使用角叉菜胶（炎症原）诱导的大鼠爪水肿模型评估 LFA-9 的抗炎功效。使用共聚焦显微镜和免疫印迹分析评估爪组织中 CD68 免疫细胞和 TNF-α 的浸润/表达。在体外使用结肠球体评估 LFA-9 的抗癌作用。
结果： LFA-9抑制mPGES-1/5-LOX及其产物PGE ₂和LTB ₄，不影响COX-1/2及其产物PGI ₂. LFA-9 与人 mPGES-1/5-LOX 酶强烈结合并诱导其二级结构的变化，从而抑制其酶活性。LFA-9 抑制大鼠角叉菜胶诱导的炎症 (70.4%) 并抑制 CD68 免疫细胞浸润 ( P ≤ 0.0001) 和 TNF-α 表达。LFA-9 通过抑制 PGE ₂ (82%) 水平在体外抑制结肠肿瘤干性 (60.2%)。
结论：总体研究结果表明，LFA-9 是一种 mPGES-1/5-LOX 双重抑制剂，具有抗炎和预防结直肠癌的活性，值得详细研究。