Marine-Derived Natural Product HDYL-GQQ-495 Targets P62 to Inhibit Autophagy.
Marine-Derived Natural Product HDYL-GQQ-495 Targets P62 to Inhibit Autophagy.
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作者：
自噬广泛涉及肿瘤、代谢和神经退行性疾病等病理生理过程，使其成为药物开发的一个有吸引力的靶点。已经开发了几种化学筛选方法来发现自噬调节化合物。然而，具有显著药理活性的海洋化合物的调节能力在很大程度上是未知的。我们利用CRISPR/Cas9敲入策略构建了EGFPKI-LC3B细胞系，其中绿色荧光指示内源性自噬调节。利用这个细胞系，我们筛选了一个由大约500种海洋天然产物和类似物组成的化合物文库，以研究改变EGFP荧光的分子。我们确定了8个潜在的增强EGFP荧光的候选菌株，其中HDYL-GQQ-495是主要的候选菌株。进一步的免疫印迹验证表明，在HDYL-GQQ-495处理后，裂解的Lc3以剂量和时间依赖的方式增加，自噬接头p62显示寡聚。我们还证明了HDYL-GQQ-495处理引起了自噬底物的聚集，这表明HDYL-GQQ-495具有自噬抑制作用。此外，HDYL-GQQ-495还可诱导Gasdermin E(GSDME)裂解并促进下垂。此外，HDYL-GQQ-495直接与p62结合，诱导p62聚合。在p62基因敲除细胞中，经HDYL-GQQ-495处理后，Lc3或GSDME的切割被阻断。EGFPKI-LC3B细胞系是筛选自噬调节子的有效工具。利用这个工具，我们发现了一种新型的海洋衍生化合物HDYL-GQQ-495，它针对p62抑制自噬和促进下垂。
Autophagy is widely implicated in pathophysiological processes such as tumors and metabolic and neurodegenerative disorders, making it an attractive target for drug discovery. Several chemical screening approaches have been developed to uncover autophagy-modulating compounds. However, the modulation capacity of marine compounds with significant pharmacological activities is largely unknown. We constructed an EGFPKI-LC3B cell line using the CRISPR/Cas9 knock-in strategy in which green fluorescence indicated endogenous autophagy regulation. Using this cell line, we screened a compound library of approximately 500 marine natural products and analogues to investigate molecules that altered the EGFP fluorescence. We identified eight potential candidates that enhanced EGFP fluorescence, and HDYL-GQQ-495 was the leading one. Further validation with immunoblotting demonstrated that cleaved LC3 was increased in dose- and time-dependent manners, and the autophagy adaptor P62 showed oligomerization after HDYL-GQQ-495 treatment. We also demonstrated that HDYL-GQQ-495 treatment caused autophagy substrate aggregation, which indicated that HDYL-GQQ-495 serves as an autophagy inhibitor. Furthermore, HDYL-GQQ-495 induced Gasdermin E (GSDME) cleavage and promoted pyroptosis. Moreover, HDYL-GQQ-495 directly combined with P62 to induce P62 polymerization. In P62 knockout cells, the cleavage of LC3 or GSDME was blocked after HDYL-GQQ-495 treatment. The EGFPKI-LC3B cell line was an effective tool for autophagy modulator screening. Using this tool, we found a novel marine-derived compound, HDYL-GQQ-495, targeting P62 to inhibit autophagy and promote pyroptosis.
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