A small-molecule activator of ULK1 that induces cytoprotective autophagy for Parkinson disease treatment

OBJECTIVE To discover a small-molecule activator of ULK1 for Parkinson disease treatment and exploreits potential mechanisms.METHODS Candidate ULK1 activator was found by using structure-based design and high-through put screening,then modified by chemical synthesis and screened by kinase and autophgic activities.The amino acid residues that key to the activation site of the best candidate ULK1 activator(BL-918) were determined by site-directed mutagenesis,as well as in vitro kinase assay,ADP-Glo kinase assay and surface plasmon resonance(SPR) analysis.The mechanisms of BL-918 induced cytoprotective autophagy were investigated by electron microscopy,fluorescence microscopy,Western blotting,co-immunoprecipitation assay,si RNA and GFP-LC3 plasmid transfections.The therapeutic effect of BL-918 was determined by MPTP-mouse model,including behavioral tests,the levels of dopamine and its derivatives,as well as immunofluorescence and Western blotting.The toxicity of BL-918 was assessed by blood sample analysis and hematoxylin-eosin staining.RESULTS We discovered a small molecule(BL-918) as a potent activator of ULK1 by structure-based drug design.Subsequently,some key amino acid residues(Arg18,Lys50,Asn86 and Tyr89) were found to be crucial to the binding pocket between ULK1 and BL-918,by site-directed mutagenesis.Moreover,we found that BL-918 could induce autophagy via the ULK complex in neuroblastoma SH-SY5Y cells.Intriguingly,this activator displayed a cytoprotective effect on MPP+-treated SH-SY5Y cells,as well as protected against MPTP-induced motor dysfunction and loss of dopaminergic neurons by targeting ULK1-modulated autophagy in mouse models of PD.CONCLUSION We discovered a novel ULK1 activator(BL-918) that potently activated ULK1.This activator could induce cytoprotective autophagy via the ULK1 complex in SH-SY5Y cells,and also exerted its neuroprotective effects by targeting ULK1-modulated autophagy in a MPTP-induced PD mouse model,which may serve as a candidate drug for future PD therapy.