ABSTRACT
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, siRNA 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.