Department of Cosmetic Science, Providence University, Taichung 433, Taiwan; Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan.
Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan.
EBioMedicine. 2019 Feb;40:528-540. doi: 10.1016/j.ebiom.2019.01.043. Epub 2019 Jan 30.
Acute lung injury (ALI) is a severe life-threatening inflammatory disease. Neutrophil activation is a major pathogenic factor in ALI. Protein kinase B (PKB)/AKT regulates diverse cellular responses, but the significance in neutrophilic inflammation and ALI remains unknown.
Human neutrophils and neutrophil-like differentiated HL-60 (dHL-60) cells were used to examine the anti-inflammatory effects of 5,7-dimethoxy-1,4-phenanthrenequinone (CLLV-1). The therapeutic potential of CLLV-1 was determined in a mouse model of lipopolysaccharide (LPS)-induced ALI.
CLLV-1 inhibited respiratory burst, degranulation, adhesion, and chemotaxis in human neutrophils and dHL-60 cells. CLLV-1 inhibited the phosphorylation of AKT (Thr308 and Ser473), but not of ERK, JNK, or p38. Furthermore, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Significantly, CLLV-1 ameliorated LPS-induced ALI, neutrophil infiltration, and AKT activation in mice.
Our results identify CLLV-1 as a covalent allosteric AKT inhibitor by targeting AKT Cys310. CLLV-1 shows potent anti-inflammatory activity in human neutrophils and LPS-induced mouse ALI. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.
急性肺损伤（ALI）是一种严重的危及生命的炎症性疾病。中性粒细胞的激活是 ALI 的主要致病因素。蛋白激酶 B（PKB）/AKT 调节多种细胞反应，但在中性粒细胞炎症和 ALI 中的意义尚不清楚。
使用人中性粒细胞和中性粒细胞样分化 HL-60（dHL-60）细胞来研究 5,7-二甲氧基-1,4-菲醌（CLLV-1）的抗炎作用。在脂多糖（LPS）诱导的 ALI 小鼠模型中确定了 CLLV-1 的治疗潜力。
CLLV-1 抑制了人中性粒细胞和 dHL-60 细胞中的呼吸爆发、脱颗粒、黏附和趋化作用。CLLV-1 抑制了 AKT（Thr308 和 Ser473）的磷酸化，但不抑制 ERK、JNK 或 p38。此外，CLLV-1 在体外阻断了 AKT 活性并与 AKT Cys310 发生共价反应。通过 NMR 或质谱测定 AKT 肽-CLLV-1 加合物。CLLV-1 还抑制 AKT 肽-烷基化剂缀合物（还原形式）水平。重要的是，CLLV-1 改善了 LPS 诱导的 ALI、小鼠中性粒细胞浸润和 AKT 激活。
我们的结果将 CLLV-1 鉴定为一种通过靶向 AKT Cys310 的共价变构 AKT 抑制剂。CLLV-1 在人中性粒细胞和 LPS 诱导的小鼠 ALI 中具有强大的抗炎活性。我们的研究结果为 AKT 的氧化还原修饰提供了一个机制框架，这可能成为减轻中性粒细胞炎症的新的药理学靶点。
