A new LKB1 activator, Piericidin Analogue S14, attenuates tubular cell senescence and renal fibrosis through promoting AMPK-induced autophagy and mitochondrial homeostasis pathway [II]

AMP-activated protein kinase (AMPK) stabilizes tubular cell metabolism and protects against renal fibrosis through promoting autophagy and mitochondrial homeostasis. Liver kinase B1 (LKB1) is the key regulator for AMPK activation. However, the direct activators of LKB1 are scarce in commercial. In this study, we report a novel LKB1 activator, the piericidin analogue S14 (PA-S14), which was isolated from the culture broth of a marine-derived Streptomyces strain by our group. PA-S14 binds with residue D176 in LKB1 kinase domain, and then induces LKB1 phosphorylated activation and its complex formation with MO25 and STRADα. Furthermore, PA-S14 promotes AMPK activation to enhance LC3B-II/LC3B-I ratio, trigger autophagosome formation and increase autophagic flux. PA-S14 exhibits perfect protective effects on stabilizing mitochondrial homeostasis, inhibiting tubular cell senescence, and retarding fibrogenesis in various CKD models (UUO, UIRI and adriamycin nephropathy models) and TGF-β-stimulated tubular cell culture. Transcriptomics sequencing and site-specific mutation analysis further prove that PA-S14 is a novel lead compound of LKB1 activator, which perfectly protects against renal fibrosis through inducing AMPK-mediated autophagy and mitochondrial homeostasis. six sample are analyzed, replicates are included, there are 3 UIRI groups and 3 UIRI+PA-S14 groups. For UIRI model, BALB/c mice were subjected to unilateral IRI. At 4 days after IRI, mice were subjected to daily intraperitoneal injections of vehicle or PA-S14 for 7 days.

腺苷酸活化蛋白激酶（AMP-activated protein kinase，AMPK）可通过促进自噬与维持线粒体稳态，维持肾小管细胞的代谢稳态并抵御肾纤维化。肝激酶B1（Liver kinase B1，LKB1）是介导AMPK激活的关键调控蛋白。然而，目前商业化的LKB1直接激活剂十分匮乏。本研究中，我们报道了一种新型LKB1激活剂——piericidin类似物S14（PA-S14），该化合物由本团队从一株海洋来源链霉菌的发酵液中分离得到。PA-S14可结合LKB1激酶结构域内的D176位点残基，进而诱导LKB1发生磷酸化激活，并促进其与MO25、STRADα形成复合物。此外，PA-S14可通过促进AMPK激活，提升LC3B-II/LC3B-I比值，诱导自噬体形成并增强自噬流。PA-S14在多种慢性肾脏病（CKD）模型（包括单侧输尿管梗阻模型UUO、缺血再灌注损伤模型UIRI以及阿霉素肾病模型）与转化生长因子-β（TGF-β）刺激的肾小管细胞培养体系中，均表现出稳定线粒体稳态、抑制肾小管细胞衰老以及延缓纤维化进程的优异保护作用。转录组测序与位点特异性突变分析进一步证实，PA-S14是一种新型LKB1激活剂先导化合物，可通过诱导AMPK介导的自噬与线粒体稳态，有效抵御肾纤维化。本研究共分析6份样本并设置生物学重复，包含3个UIRI模型组与3个UIRI+PA-S14给药组。针对UIRI模型，实验采用BALB/c小鼠构建单侧缺血再灌注损伤模型；于造模后第4天，每日对小鼠进行腹腔注射溶剂对照或PA-S14，连续给药7天。