Aim Recent evidence has revealed that Eukaryotic elongation factor-2 kinase （eEF2K） activity may confer cancer cell adaptation to metabolic stress, and high expression of eEF2K is found in several types of cancer. T...Aim Recent evidence has revealed that Eukaryotic elongation factor-2 kinase （eEF2K） activity may confer cancer cell adaptation to metabolic stress, and high expression of eEF2K is found in several types of cancer. Therefore, eEF2K may contribute to carcinogenesis and represent a promising therapeutic target; however, inhibi- tion of eEF2K for cancer drug discovery still remains in its infancy. This study aimed at developing a series of eEF2K inhibitor as candidate anti-tumor drugs in breast cancer and illustrating the possible mechanisms of its anti- tumor activity in vitro and in vivo. Methods In silico screening, structure modifications, MTT assay and molecular dynamics （MD） simulations were applied for the discovery of the novel eEF2K inhibitor （BL-EKI03）. Observa- tions of cell morphology were executed through several methods including ER-traeker, MDC and Hoeehst 33258 staining and GFP-LC3 transfeetion. Flow eytometrie analyses of MDC and Annexin V/PI were used for quantifica- tion of autophagy and apoptosis ratio. Western blot and ITRAQ analysis were used to explore the detailed mecha- nisms of BL-EKI03-induced ER stress, autophagie death and apoptosis in breast cancer cells. Furthermore, an in vivo xenograft mouse model was established for validating the anti-tumor efficacy of BL-EKI03. Results Firstly, a novel eEF2K inhibitor （BL-EKI03） with a good affinity for eEF2K was eventually discovered after computational screening and synthesis of a series of candidate compounds targeting eEF2K. Subsequently, our results demonstra- ted that BL-EKI03 has remarkable anti-proliferative activities and induces endoplasmie retieulum （ER） stress, au- tophagy and apoptosis in MCF-7 and MDA-MB-436 cells. More importantly, the mechanism for BL-EKI03-indueed autophagie death involves eEF2K-mediated AMPK-mTOR-ULK complex pathways. The proteomies analyses and ex-perimental validation revealed that the BL-EKI03-induced mechanism was also involved BIRC6, BNIP1, SNAP29 and Bif-1, which might be regulated by eEF2K. Moreover, BL-EKI03 exerted its anti-tumor activities without re- markable toxicity, and it also induced autophagy and apoptosis by targeting eEF2K in fifo. Conclusion In this study, a novel eEF2K inhibitor （BL-EKI03） was discovered with remarkable anti-proliferative activities and in- duced endoplasmic reticulum （ER） stress, autophagy and apoptosis of breast cancer in vitro and in fifo. These findings highlight a new small-molecule eEF2K inhibitor （BL-EKI03） that has the potential to impact future breast cancer therapy.展开更多
Chronic kidney disease(CKD)affects 8%e15%of the population globally and can cause renal failure,partly due to lack of effective treatments and drug targets.Three novel cembrane diterpenoids papyifurans A‒C(1e3),in par...Chronic kidney disease(CKD)affects 8%e15%of the population globally and can cause renal failure,partly due to lack of effective treatments and drug targets.Three novel cembrane diterpenoids papyifurans A‒C(1e3),in particular of 1 with an unprecedented trioxatetracyclo[10.2.1.1^(2,5).1^(6,9)]heptadecane polyether scaffold,derived from Boswellia papyrifera resin,were found to effectively protect against renal fibrosis in vitro and in vivo.Their structures were fully characterized using a combination of spectroscopic,computational,modified Mosher’s,and X-ray crystallographic analysis.In particular,we performed chemical proteomic analyses and found that Elongation factor 2(EEF2)is the key target of compound 1 for anti-renal fibrosis in vitro.Moreover,previous studies have linked EEF2 with lung fibrosis,while compound 1 was found to inhibit the hallmarks of organ fibrosis in vitro.Such effects were observed to decrease with the knock down of EEF2 in vitro,suggesting that EEF2 might be a universal drug target of organ fibrosis.Collectively,the present study demonstrated an example of identifying drug targets by using structurally novel natural products,which will be beneficial for developing therapeutic agents against organ fibrosis.展开更多
TOR(target of rapamycin)是真核生物中高度保守的一种大分子的Ser/Thr激酶,是免疫抑制剂雷帕霉素的在体内的靶物质。TOR能够对营养状况和生长因子等因素的变化做出应答反应,通过介导磷酸化反应调节蛋白激酶4E-BP1,S6K, eEF2和磷酸酶等...TOR(target of rapamycin)是真核生物中高度保守的一种大分子的Ser/Thr激酶,是免疫抑制剂雷帕霉素的在体内的靶物质。TOR能够对营养状况和生长因子等因素的变化做出应答反应,通过介导磷酸化反应调节蛋白激酶4E-BP1,S6K, eEF2和磷酸酶等的活性,控制下游翻译因子的磷酸化水平,调节核糖体发生,蛋白质合成等生理过程,在细胞的生长,增殖的综合调控中起到中枢作用。展开更多
Objective:Understanding of the molecular mechanisms underlying age-associated cognitive impairments will not only contribute to our general knowledge about“aging”biology,but also provide insights for more effective ...Objective:Understanding of the molecular mechanisms underlying age-associated cognitive impairments will not only contribute to our general knowledge about“aging”biology,but also provide insights for more effective strategies to prevent and improve the quality of life for both normal aging and pathological aging such as Alzheimer’s disease(AD).multiple lines of evidence suggest that subtle morphological and/or biochemical neuronal changes,instead of profound loss of neurons,is responsible for aging-related impairments of cognition and synaptic plasticity,which is often measured in vertebrates as long-term potentiation(LTP),a synaptic model for memory.A substantial body of evidence demonstrates that de novo protein synthesis(mRNA translation)is indispensable to maintain long-lasting forms of memory and synaptic plasticity.Of interest,activities of translational factors involved into various stages of protein synthesis and synthesis of translational machinery per se(i.e.translational capacity)are known to be regulated in synaptic plasticity and memory formation by various signaling pathways.Methods:Breeders for C57 BL/6 J mice were purchased from the Jackson Laboratory(Bar Harbor,ME USA).All mice were housed in the barrier Mouse Facility at Wake Forest School of Medicine Animal Facility.Mice were kept in compliance with the National Institute of Health(NIH)guide for Care and Use of Laboratory Animals.The facility kept a 12 hour light/dark cycle with regular feeding,cage cleaning,and 24 hour access to water.Male and female mice,aged 3~5 or 19~21 months,were used for these experiments.Mice were subjected to the following behavioral tasks:hidden platform Morris water maze,which was consisted of 4 trials each day for 5 consecutive days,with probe trial being carried out 2 hours after training day 5;visible platform task,which was consisted of 4 trials each day for 2 consecutive days with the escape platform marked by a visible cue and moves randomly between four locations;novel object recognition,in which the amount of time spent exploring the novel object was normalized by the total time spent exploring both objects to yield a preference index to calculate percent object preference;reversal Y water maze:mice were trained to pick up one side of the maze,where a platform was hidden.The memory test phase began after a delay of 24 hours,which included 5 trials.For mice chose the right arm,the escape platform was switched to the opposite arm,and the mice were trained to learn the new location of the platform.For electrophysiology experiments,acute 400μm transverse hippocampal slices were prepared using a Leica VT1200S vibratome.Late long-term potentiation(LTP)was induced using high-frequency stimulation(HFS)consisting of two 1-sec 100 Hz trains separated by 60 sec,each delivered at 70%~80%of the intensity that evoked spiked fEPSPs.Early LTP was induced using one-train HFS(100 Hz)delivered at 25%~30%of the intensity that evoked spiked fEPSPs.Mouse brain tissue was harvested for biochemical experiments as described before(Ma et al.,Nature Neuroscience,2013).Results:Here we fi rst assessed and compared the performance of cognition and synaptic plasticity in young(3~5 month old)and aged c57 BL/6 J mice(19~21 months old).Findings from behavioral tests demonstrated that old mice,compared to young mice,displayed impairments in spatial learning/memory,working memory,and behavioral fl exibility.Further,synaptic electrophysiology experiments on hippocampal slices revealed that early form LTP(a synaptic model for memory formation)was inhibited in old mice.At the molecular level,biochemical assays on brain tissue showed dysregulation of signaling pathways controlling protein synthesis capacity including:up-regulation of AKT-mTORC1-p70S6K signaling,which is associated with translation of terminal oligopyrimidine(TOP)class of mRNAs that encode translational machinery;hyper-phosphorylation of mRNA translational elongation factor 2(eEF2)and its upstream regulator AMP-activated protein kinase(AMPK),indicating repression of general protein synthesis.Moreover,young and old mice exhibited similar brain levels of translational initiation factor 2α(eIF2α)phosphorylation,which is known to be increased in AD and linked to the disease pathophysiology.Conclusion:Our fi ndings provide evidence at the molecular level to highlight the similarity and difference between normal and pathological aging,which may contribute to future studies on diagnostic/prognostic biomarkers for aging-related dementia syndromes.展开更多
文摘Aim Recent evidence has revealed that Eukaryotic elongation factor-2 kinase （eEF2K） activity may confer cancer cell adaptation to metabolic stress, and high expression of eEF2K is found in several types of cancer. Therefore, eEF2K may contribute to carcinogenesis and represent a promising therapeutic target; however, inhibi- tion of eEF2K for cancer drug discovery still remains in its infancy. This study aimed at developing a series of eEF2K inhibitor as candidate anti-tumor drugs in breast cancer and illustrating the possible mechanisms of its anti- tumor activity in vitro and in vivo. Methods In silico screening, structure modifications, MTT assay and molecular dynamics （MD） simulations were applied for the discovery of the novel eEF2K inhibitor （BL-EKI03）. Observa- tions of cell morphology were executed through several methods including ER-traeker, MDC and Hoeehst 33258 staining and GFP-LC3 transfeetion. Flow eytometrie analyses of MDC and Annexin V/PI were used for quantifica- tion of autophagy and apoptosis ratio. Western blot and ITRAQ analysis were used to explore the detailed mecha- nisms of BL-EKI03-induced ER stress, autophagie death and apoptosis in breast cancer cells. Furthermore, an in vivo xenograft mouse model was established for validating the anti-tumor efficacy of BL-EKI03. Results Firstly, a novel eEF2K inhibitor （BL-EKI03） with a good affinity for eEF2K was eventually discovered after computational screening and synthesis of a series of candidate compounds targeting eEF2K. Subsequently, our results demonstra- ted that BL-EKI03 has remarkable anti-proliferative activities and induces endoplasmie retieulum （ER） stress, au- tophagy and apoptosis in MCF-7 and MDA-MB-436 cells. More importantly, the mechanism for BL-EKI03-indueed autophagie death involves eEF2K-mediated AMPK-mTOR-ULK complex pathways. The proteomies analyses and ex-perimental validation revealed that the BL-EKI03-induced mechanism was also involved BIRC6, BNIP1, SNAP29 and Bif-1, which might be regulated by eEF2K. Moreover, BL-EKI03 exerted its anti-tumor activities without re- markable toxicity, and it also induced autophagy and apoptosis by targeting eEF2K in fifo. Conclusion In this study, a novel eEF2K inhibitor （BL-EKI03） was discovered with remarkable anti-proliferative activities and in- duced endoplasmic reticulum （ER） stress, autophagy and apoptosis of breast cancer in vitro and in fifo. These findings highlight a new small-molecule eEF2K inhibitor （BL-EKI03） that has the potential to impact future breast cancer therapy.
基金supported by Shenzhen Fundamental Research Program(No.JCYJ20200109114003921,China).
文摘Chronic kidney disease(CKD)affects 8%e15%of the population globally and can cause renal failure,partly due to lack of effective treatments and drug targets.Three novel cembrane diterpenoids papyifurans A‒C(1e3),in particular of 1 with an unprecedented trioxatetracyclo[10.2.1.1^(2,5).1^(6,9)]heptadecane polyether scaffold,derived from Boswellia papyrifera resin,were found to effectively protect against renal fibrosis in vitro and in vivo.Their structures were fully characterized using a combination of spectroscopic,computational,modified Mosher’s,and X-ray crystallographic analysis.In particular,we performed chemical proteomic analyses and found that Elongation factor 2(EEF2)is the key target of compound 1 for anti-renal fibrosis in vitro.Moreover,previous studies have linked EEF2 with lung fibrosis,while compound 1 was found to inhibit the hallmarks of organ fibrosis in vitro.Such effects were observed to decrease with the knock down of EEF2 in vitro,suggesting that EEF2 might be a universal drug target of organ fibrosis.Collectively,the present study demonstrated an example of identifying drug targets by using structurally novel natural products,which will be beneficial for developing therapeutic agents against organ fibrosis.
文摘TOR(target of rapamycin)是真核生物中高度保守的一种大分子的Ser/Thr激酶,是免疫抑制剂雷帕霉素的在体内的靶物质。TOR能够对营养状况和生长因子等因素的变化做出应答反应,通过介导磷酸化反应调节蛋白激酶4E-BP1,S6K, eEF2和磷酸酶等的活性,控制下游翻译因子的磷酸化水平,调节核糖体发生,蛋白质合成等生理过程,在细胞的生长,增殖的综合调控中起到中枢作用。
文摘Objective:Understanding of the molecular mechanisms underlying age-associated cognitive impairments will not only contribute to our general knowledge about“aging”biology,but also provide insights for more effective strategies to prevent and improve the quality of life for both normal aging and pathological aging such as Alzheimer’s disease(AD).multiple lines of evidence suggest that subtle morphological and/or biochemical neuronal changes,instead of profound loss of neurons,is responsible for aging-related impairments of cognition and synaptic plasticity,which is often measured in vertebrates as long-term potentiation(LTP),a synaptic model for memory.A substantial body of evidence demonstrates that de novo protein synthesis(mRNA translation)is indispensable to maintain long-lasting forms of memory and synaptic plasticity.Of interest,activities of translational factors involved into various stages of protein synthesis and synthesis of translational machinery per se(i.e.translational capacity)are known to be regulated in synaptic plasticity and memory formation by various signaling pathways.Methods:Breeders for C57 BL/6 J mice were purchased from the Jackson Laboratory(Bar Harbor,ME USA).All mice were housed in the barrier Mouse Facility at Wake Forest School of Medicine Animal Facility.Mice were kept in compliance with the National Institute of Health(NIH)guide for Care and Use of Laboratory Animals.The facility kept a 12 hour light/dark cycle with regular feeding,cage cleaning,and 24 hour access to water.Male and female mice,aged 3~5 or 19~21 months,were used for these experiments.Mice were subjected to the following behavioral tasks:hidden platform Morris water maze,which was consisted of 4 trials each day for 5 consecutive days,with probe trial being carried out 2 hours after training day 5;visible platform task,which was consisted of 4 trials each day for 2 consecutive days with the escape platform marked by a visible cue and moves randomly between four locations;novel object recognition,in which the amount of time spent exploring the novel object was normalized by the total time spent exploring both objects to yield a preference index to calculate percent object preference;reversal Y water maze:mice were trained to pick up one side of the maze,where a platform was hidden.The memory test phase began after a delay of 24 hours,which included 5 trials.For mice chose the right arm,the escape platform was switched to the opposite arm,and the mice were trained to learn the new location of the platform.For electrophysiology experiments,acute 400μm transverse hippocampal slices were prepared using a Leica VT1200S vibratome.Late long-term potentiation(LTP)was induced using high-frequency stimulation(HFS)consisting of two 1-sec 100 Hz trains separated by 60 sec,each delivered at 70%~80%of the intensity that evoked spiked fEPSPs.Early LTP was induced using one-train HFS(100 Hz)delivered at 25%~30%of the intensity that evoked spiked fEPSPs.Mouse brain tissue was harvested for biochemical experiments as described before(Ma et al.,Nature Neuroscience,2013).Results:Here we fi rst assessed and compared the performance of cognition and synaptic plasticity in young(3~5 month old)and aged c57 BL/6 J mice(19~21 months old).Findings from behavioral tests demonstrated that old mice,compared to young mice,displayed impairments in spatial learning/memory,working memory,and behavioral fl exibility.Further,synaptic electrophysiology experiments on hippocampal slices revealed that early form LTP(a synaptic model for memory formation)was inhibited in old mice.At the molecular level,biochemical assays on brain tissue showed dysregulation of signaling pathways controlling protein synthesis capacity including:up-regulation of AKT-mTORC1-p70S6K signaling,which is associated with translation of terminal oligopyrimidine(TOP)class of mRNAs that encode translational machinery;hyper-phosphorylation of mRNA translational elongation factor 2(eEF2)and its upstream regulator AMP-activated protein kinase(AMPK),indicating repression of general protein synthesis.Moreover,young and old mice exhibited similar brain levels of translational initiation factor 2α(eIF2α)phosphorylation,which is known to be increased in AD and linked to the disease pathophysiology.Conclusion:Our fi ndings provide evidence at the molecular level to highlight the similarity and difference between normal and pathological aging,which may contribute to future studies on diagnostic/prognostic biomarkers for aging-related dementia syndromes.
