The PIK3C3/VPS34-containing phosphatidylinositol 3-kinase (PtdIns3K) initiation complex (complex I) is necessary for macroautophagy/autophagy initiation and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-ATG14, while the endosomal trafficking complex (complex II) is necessary for vesicle trafficking and is comprised of PIK3R4/VPS15-PIK3C3/VPS34-BECN1-UVRAG. This composition difference was exploited to identify novel and specific autophagy inhibitors that disrupted the BECN1-ATG14 protein-protein interaction, without affecting vesicle trafficking. A cellular NanoBRET assay was implemented to identify these inhibitors, and one compound was able to successfully disrupt the BECN1-ATG14 interaction and inhibit autophagy, with limited impact on vesicle trafficking. These results reveal the first protein-protein interaction inhibitor targeting the autophagy initiation machinery and demonstrate the viability of targeting protein-protein interactions for the discovery of autophagy-specific modulators.

包含PIK3C3 / VPS34的磷脂酰肌醇3-激酶（PtdIns3K）起始复合物（复合物I）对于巨噬自噬/自噬起始是必不可少的，并且由PIK3R4 / VPS15-PIK3C3 / VPS34-BECN1-ATG14组成，而内体运输复合物（复合物II） ）是囊泡运输所必需的，且由PIK3R4 / VPS15-PIK3C3 / VPS34-BECN1-UVRAG组成。利用这种成分差异来鉴定新颖且特异性的自噬抑制剂，该抑制剂可破坏BECN1-ATG14蛋白质-蛋白质相互作用，而不会影响囊泡运输。实施了细胞NanoBRET分析以鉴定这些抑制剂，并且一种化合物能够成功破坏BECN1-ATG14相互作用并抑制自噬，但对囊泡运输的影响有限。