OBJECTIVE To discover a small molecule targeting ULK1-modulated cell death of triple negative breast cancer and exploreits potential mechanisms.METHODS ULK1 expression was analyzed by The Cancer Genome Atlas(TCGA)anal...OBJECTIVE To discover a small molecule targeting ULK1-modulated cell death of triple negative breast cancer and exploreits potential mechanisms.METHODS ULK1 expression was analyzed by The Cancer Genome Atlas(TCGA)analysis and tissue microarray(TMA)analysis.ULK1agonist was designed by using in silico screening,as well as modified by chemical synthesis and screened by kinase and anti-proliferative activities.The amino acid residues that key to the activation site of LYN-1604 were determined by site-directed mutagenesis,as well as in vitro kinase assay and ADP-Glo kinase assay.The mechanisms of LYN-1604 induced cell death were investigated by fluorescence microscope,western blotting,flow cytometry analysis,immunocytochemistry,as well as si RNA and GFP-m RFP-LC3 plasmid transfections.Potential ULK1 interactors were discovered by performing comparative microarray analysis and the therapeutic effect of LYN-1604 was assessed by xenograft breast cancer mouse model.RESULTS We found that ULK1 was remarkably downregulated in breast cancer tissue samples,especial y in triple negative breast cancer(TNBC).32 candidate smal molecules were synthesized,and we discovered a small molecule named LYN-1604 as the best candidate ULK1agonist.Additionally,we identified that three amino acid residues(LYS50,LEU53 and TYR89)were key to the activation site of LYN-1604 and ULK1.Subsequently,we demonstrated that LYN-1604 could induce autophagy-associated cell death via ULK complex(ULK1-m ATG13-FIP200-ATG101)in MDA-MB-231 cells.We also found that LYN-1604 induced cell death involved in ATF3,RAD21 and caspase 3,accompanied with autophagy and apoptosis.Moreover,we demonstrated that LYN-1604 had a good therapeutic potential on TNBC by targeting ULK1-modulated cell death in vivo.CONCLUSION We discovered a small molecule(LYN-1604)has therapeutic potential by targeting ULK1-modulated cell death associated with autophagy and apoptosis of TNBC in vitro and in vivo,which could be utilized as a new anti-TNBC drug candidate.展开更多
UNC-51-like kinase 1(ULK1),as a serine/threonine kinase,is an autophagic initiator in mammals and a homologous protein of autophagy related protein(Atg)1 in yeast and of UNC-51 in Caenorhabditis elegans.ULK1 is well-k...UNC-51-like kinase 1(ULK1),as a serine/threonine kinase,is an autophagic initiator in mammals and a homologous protein of autophagy related protein(Atg)1 in yeast and of UNC-51 in Caenorhabditis elegans.ULK1 is well-known for autophagy activation,which is evolutionarily conserved in protein transport and indispensable to maintain cell homeostasis.As the direct target of energy and nutrition-sensing kinase,ULK1 may contribute to the distribution and utilization of cellular resources in response to metabolism and is closely associated with multiple pathophysiological processes.Moreover,ULK1 has been widely reported to play a crucial role in human diseases,including cancer,neurodegenerative diseases,cardiovascular disease,and infections,and subsequently targeted small-molecule inhibitors or activators are also demonstrated.Interestingly,the non-autophagy function of ULK1 has been emerging,indicating that non-autophagy-relevant ULK1 signaling network is also linked with diseases under some specific contexts.Therefore,in this review,we summarized the structure and functions of ULK1 as an autophagic initiator,with a focus on some new approaches,and further elucidated the key roles of ULK1 in autophagy and non-autophagy.Additionally,we also discussed the relationships between ULK1 and human diseases,as well as illustrated a rapid progress for better understanding of the discovery of more candidate small-molecule drugs targeting ULK1,which will provide a clue on novel ULK1-targeted therapeutics in the future.展开更多
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 ...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.展开更多
Dear Editor,Macroautophagy(referred to as autophagy herein)is an evolutionarily con served,lysosomal degradatio n process by which cells rid themselves of aggregated proteins and damaged organelles(He and Klionsky,200...Dear Editor,Macroautophagy(referred to as autophagy herein)is an evolutionarily con served,lysosomal degradatio n process by which cells rid themselves of aggregated proteins and damaged organelles(He and Klionsky,2009).This process involves a finely orchestrated molecular pathway comprising a plethora of ATG proteins essential for autophagosome formation.The mammalian Unc-51-like kinase(ULK)complex plays an essential role to initiate canonical autophagy pathway by relaying upstream nutrient and stress signals to downstream autophagy machinery(Mizushima,2010;Wong et al.,2013).展开更多
Purpose:To observe the expression differences and potential effects of autophagy-related Beclin1(mammalian Atg6)and Uncoordinated-51 like kinase 1(ULK1)in the oxygen-induced retinopathy(OIR)model.Materials and methods...Purpose:To observe the expression differences and potential effects of autophagy-related Beclin1(mammalian Atg6)and Uncoordinated-51 like kinase 1(ULK1)in the oxygen-induced retinopathy(OIR)model.Materials and methods:Thirty-three C57BL/6 mice in OIR model group were exposed to 750.5% oxygen from postnatal day-of-life 7(P7)to P12,and were then brought into normal room environment(relative hypoxia stage)and raised to P17.Thirty-three control mice were kept in a normal room environment.The expression of autophagy in the retina tissue was assessed by Western blot analysis.The thickness and ultrastructural of retina were observed by light microscopy and transmission electron microscope(TEM)on P17.Results:In the hyperoxia stage(P8–P11),the expression of Beclin1,ULK1 and Autophagy 5(Atg5)in retina showed no significant difference between the OIR model group and the control group.In the relatively hypoxia stage(P14 to P17),however,the protein level of Beclin1,ULK1,and Bcl-2-associated X protein(Bax)were upregulated in the retina of the OIR model group,whereas B-cell lymphoma 2(Bcl-2)was downregulated.The autophagosomes in the photoreceptors of retina in the OIR mice were observed.The inner-segment/out-segment(IS/OS)layer in OIR model group was thinner than that the control group on P17.Conclusions:The expression of Beclin-1 and ULK1 in retina has changed in the OIR model,and the change of Beclin-1 and ULK1 expression is related to the change of oxygen concentration.展开更多
基金supported by National Natural Science Foundation of China(81402496,81673455and 81602627)China Postdoctoral Special Science Foundation(2017T100704)China Postdoctoral Science Foundation(2015M580794)
文摘OBJECTIVE To discover a small molecule targeting ULK1-modulated cell death of triple negative breast cancer and exploreits potential mechanisms.METHODS ULK1 expression was analyzed by The Cancer Genome Atlas(TCGA)analysis and tissue microarray(TMA)analysis.ULK1agonist was designed by using in silico screening,as well as modified by chemical synthesis and screened by kinase and anti-proliferative activities.The amino acid residues that key to the activation site of LYN-1604 were determined by site-directed mutagenesis,as well as in vitro kinase assay and ADP-Glo kinase assay.The mechanisms of LYN-1604 induced cell death were investigated by fluorescence microscope,western blotting,flow cytometry analysis,immunocytochemistry,as well as si RNA and GFP-m RFP-LC3 plasmid transfections.Potential ULK1 interactors were discovered by performing comparative microarray analysis and the therapeutic effect of LYN-1604 was assessed by xenograft breast cancer mouse model.RESULTS We found that ULK1 was remarkably downregulated in breast cancer tissue samples,especial y in triple negative breast cancer(TNBC).32 candidate smal molecules were synthesized,and we discovered a small molecule named LYN-1604 as the best candidate ULK1agonist.Additionally,we identified that three amino acid residues(LYS50,LEU53 and TYR89)were key to the activation site of LYN-1604 and ULK1.Subsequently,we demonstrated that LYN-1604 could induce autophagy-associated cell death via ULK complex(ULK1-m ATG13-FIP200-ATG101)in MDA-MB-231 cells.We also found that LYN-1604 induced cell death involved in ATF3,RAD21 and caspase 3,accompanied with autophagy and apoptosis.Moreover,we demonstrated that LYN-1604 had a good therapeutic potential on TNBC by targeting ULK1-modulated cell death in vivo.CONCLUSION We discovered a small molecule(LYN-1604)has therapeutic potential by targeting ULK1-modulated cell death associated with autophagy and apoptosis of TNBC in vitro and in vivo,which could be utilized as a new anti-TNBC drug candidate.
基金supported in part by National Natural Science Foundation of China (Grant Nos. 82172649 and 82173666)Shenzhen science and technology research and development funds (Grant No. JCYJ20210324094612035, China)the Key R&D Program of Sichuan Province (Grant No. 2021YFS0046, China)
文摘UNC-51-like kinase 1(ULK1),as a serine/threonine kinase,is an autophagic initiator in mammals and a homologous protein of autophagy related protein(Atg)1 in yeast and of UNC-51 in Caenorhabditis elegans.ULK1 is well-known for autophagy activation,which is evolutionarily conserved in protein transport and indispensable to maintain cell homeostasis.As the direct target of energy and nutrition-sensing kinase,ULK1 may contribute to the distribution and utilization of cellular resources in response to metabolism and is closely associated with multiple pathophysiological processes.Moreover,ULK1 has been widely reported to play a crucial role in human diseases,including cancer,neurodegenerative diseases,cardiovascular disease,and infections,and subsequently targeted small-molecule inhibitors or activators are also demonstrated.Interestingly,the non-autophagy function of ULK1 has been emerging,indicating that non-autophagy-relevant ULK1 signaling network is also linked with diseases under some specific contexts.Therefore,in this review,we summarized the structure and functions of ULK1 as an autophagic initiator,with a focus on some new approaches,and further elucidated the key roles of ULK1 in autophagy and non-autophagy.Additionally,we also discussed the relationships between ULK1 and human diseases,as well as illustrated a rapid progress for better understanding of the discovery of more candidate small-molecule drugs targeting ULK1,which will provide a clue on novel ULK1-targeted therapeutics in the future.
基金supported by National Natural Science Foundation of China(81602953)China Postdoctoral Special Science Foundation(2017T100706)China Postdoctoral Science Foundation(2016M590893)
文摘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.
文摘Dear Editor,Macroautophagy(referred to as autophagy herein)is an evolutionarily con served,lysosomal degradatio n process by which cells rid themselves of aggregated proteins and damaged organelles(He and Klionsky,2009).This process involves a finely orchestrated molecular pathway comprising a plethora of ATG proteins essential for autophagosome formation.The mammalian Unc-51-like kinase(ULK)complex plays an essential role to initiate canonical autophagy pathway by relaying upstream nutrient and stress signals to downstream autophagy machinery(Mizushima,2010;Wong et al.,2013).
基金supported by grants from the Natural Science Foundation of Zhejiang Province,(NO.LQ18H120003)Foundation of Zhejiang Medical and Health Science and Technology Project,(NO.2018KY554).
文摘Purpose:To observe the expression differences and potential effects of autophagy-related Beclin1(mammalian Atg6)and Uncoordinated-51 like kinase 1(ULK1)in the oxygen-induced retinopathy(OIR)model.Materials and methods:Thirty-three C57BL/6 mice in OIR model group were exposed to 750.5% oxygen from postnatal day-of-life 7(P7)to P12,and were then brought into normal room environment(relative hypoxia stage)and raised to P17.Thirty-three control mice were kept in a normal room environment.The expression of autophagy in the retina tissue was assessed by Western blot analysis.The thickness and ultrastructural of retina were observed by light microscopy and transmission electron microscope(TEM)on P17.Results:In the hyperoxia stage(P8–P11),the expression of Beclin1,ULK1 and Autophagy 5(Atg5)in retina showed no significant difference between the OIR model group and the control group.In the relatively hypoxia stage(P14 to P17),however,the protein level of Beclin1,ULK1,and Bcl-2-associated X protein(Bax)were upregulated in the retina of the OIR model group,whereas B-cell lymphoma 2(Bcl-2)was downregulated.The autophagosomes in the photoreceptors of retina in the OIR mice were observed.The inner-segment/out-segment(IS/OS)layer in OIR model group was thinner than that the control group on P17.Conclusions:The expression of Beclin-1 and ULK1 in retina has changed in the OIR model,and the change of Beclin-1 and ULK1 expression is related to the change of oxygen concentration.
文摘【目的】明确Src与胶原同源插头蛋白(Src homology and collagen homology,Shc)调控曲格列酮(troglitazone,TZ)引起的猪血管内皮(porcine aortic endothelial,PAE)细胞自噬的机制。【方法】我们先利用激光共聚焦显微镜、蛋白免疫杂交检测了TZ引起的PAE细胞自噬;然后通过siRNA干扰敲降Shc,转染wtShc、3mShc等质粒的方法确定了p52Shc参与自噬的调控;最后通过siRNA干扰敲降Ulk1得到最终结论。【结果】通过研究发现,敲降Shc,会增加细胞的自噬;而过量表达p52Shc抑制了TZ引起的细胞自噬;p52Shc抑制自噬与其自身的酪氨酸磷酸化位点Tyr239、Tyr240和Tyr317相关;同时发现,p52Shc能抑制自噬调节分子磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase,AMPK)及其下游底物Unc51样激酶1(UNC-51-like kinase-1,Ulk1)的活性。【结论】Shc通过调控AMPK与Ulk1的磷酸化调节TZ引起的细胞自噬。
