Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine(CQ) has played an i...Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine(CQ) has played an indispensable role, however, its mechanism of action(MoA) is not fully understood.Methods: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling(ABPP) and mass spectrometry-coupled cellular thermal shift assay(MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays.Results: We developed a novel clickable, photo-affinity chloroquine analog probe(CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photocrosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods.Conclusions: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled(ABPP)and label-free(MS-CETSA) methods.展开更多
活性天然产物的作用靶标、具体作用机制难以确定,阻碍了其在新药研发方面的发展。化学蛋白质组学,特别是基于亲和性蛋白组学分析方法(affinity-based protein profiling,ABPP),经过近年的发展,已成为较为成熟的鉴定天然产物作用靶标的...活性天然产物的作用靶标、具体作用机制难以确定,阻碍了其在新药研发方面的发展。化学蛋白质组学,特别是基于亲和性蛋白组学分析方法(affinity-based protein profiling,ABPP),经过近年的发展,已成为较为成熟的鉴定天然产物作用靶标的方法。本文主要以天然产物结构进行分类介绍了基于亲和性蛋白组学分析方法(affinity-based protein profiling,ABPP)在天然产物作用靶标鉴定方面的应用,并讨论了各种探针设计的应用范围以及优缺点,且对ABPP方法的发展应用和其他靶标蛋白鉴定方法进行展望。展开更多
活性蛋白质表达谱(activity-based protein profiling,ABPP)分析技术是功能蛋白质组学的一种策略,属于化学蛋白质组学的一部分.它借助化学小分子从功能角度直接切入蛋白质组的研究,能够直接对蛋白质组中感兴趣的靶酶蛋白的活性进行检测...活性蛋白质表达谱(activity-based protein profiling,ABPP)分析技术是功能蛋白质组学的一种策略,属于化学蛋白质组学的一部分.它借助化学小分子从功能角度直接切入蛋白质组的研究,能够直接对蛋白质组中感兴趣的靶酶蛋白的活性进行检测,为药物的发现提供强有力的支持.因此,ABPP技术被认为是基于功能的新一代蛋白质组学技术.随着ABPP分析技术和方法的不断成熟,其应用领域也不断扩展.最近一系列研究表明,今后ABPP分析技术可能成为病毒学研究的又一重要武器.本文综述了ABPP分析技术的基本原理及其在病毒学研究中的应用.展开更多
Schisandrin A is a natural dibenzocyclooctene lignan with potent neuroprotective activity.However,the specific mechanisms or direct target proteins have not been clarified up to now.In this study,we designed and synth...Schisandrin A is a natural dibenzocyclooctene lignan with potent neuroprotective activity.However,the specific mechanisms or direct target proteins have not been clarified up to now.In this study,we designed and synthesized the probes of schisandrin A with photoreactive diazirine and clickable alkyne to identify its direct target in SH-SY5Y cells by employing activity-based protein profiling(ABPP)technique.Ykt6 was prominent among the 13 proteins obtained with high confidence and we confirmed Ykt6 as the direct target of schisandrin A by CETSA,IF,SPR and knockdown assay.Functionally,schisandrin A protected the cells against the injury induced by glutamate by regulating autophagy via Ykt6.This discovery may provide a novel therapeutic option for various neuronal cell damage-mediated diseases.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
Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron-and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfor...Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron-and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species(ROS) and inducing ferroptosis in activated hepatic stellate cells(HSCs). By using activity-based protein profiling(ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay(CETSA), we show that celastrol directly binds to peroxiredoxins(PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6,through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1(HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2,PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis.展开更多
Seven photoaffinity-based and sixteen biotin-based berberine(BBR)probes were constructed and screened for their effects on c-Jun N-terminal protein kinases(JNK)phosphorylation(p-JNK)suppression at the cellular level.T...Seven photoaffinity-based and sixteen biotin-based berberine(BBR)probes were constructed and screened for their effects on c-Jun N-terminal protein kinases(JNK)phosphorylation(p-JNK)suppression at the cellular level.Taking active-photoaffinity probe 7c as a chemical tool,we first identified mitogen-activated protein kinase 7(MAP2K7),an upstream protein on the JNK/stress activated protein kinase(SAPK)pathway,as a direct proteomic target of BBR using activity-based protein profiling(ABPP)and other chemical proteomic techniques.Furthermore,BBR’s inhibitory effect on p-JNK was significantly attenuated in both the MAP2K7-knockdown and models,indicating a MAP2K7-dependent inhibition on the JNK signaling pathway.For the first time,we demonstrate the unique mechanism of BBR that directly targets MAP2K7 to inhibit p-JNK rather than JNK activity with the advantages of multiple activities and a good safety profile.展开更多
基金suppor ted by the National Key Research and Development Program of China(2020YFA0908000)the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(ZYYCXTD-C-202002)+2 种基金the National Natural Science Foundation of China(82074098,82003814)the CACMS Innovation Fund(CI2021A05101)the Fundamental Research Funds for the Central public welfare research institutes(ZZ14-YQ-050,ZZ14-YQ-051,ZZ14-ND-010,ZZ15-ND-10 and ZZ14-FL-002)。
文摘Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine(CQ) has played an indispensable role, however, its mechanism of action(MoA) is not fully understood.Methods: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling(ABPP) and mass spectrometry-coupled cellular thermal shift assay(MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays.Results: We developed a novel clickable, photo-affinity chloroquine analog probe(CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photocrosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods.Conclusions: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled(ABPP)and label-free(MS-CETSA) methods.
文摘活性天然产物的作用靶标、具体作用机制难以确定,阻碍了其在新药研发方面的发展。化学蛋白质组学,特别是基于亲和性蛋白组学分析方法(affinity-based protein profiling,ABPP),经过近年的发展,已成为较为成熟的鉴定天然产物作用靶标的方法。本文主要以天然产物结构进行分类介绍了基于亲和性蛋白组学分析方法(affinity-based protein profiling,ABPP)在天然产物作用靶标鉴定方面的应用,并讨论了各种探针设计的应用范围以及优缺点,且对ABPP方法的发展应用和其他靶标蛋白鉴定方法进行展望。
文摘活性蛋白质表达谱(activity-based protein profiling,ABPP)分析技术是功能蛋白质组学的一种策略,属于化学蛋白质组学的一部分.它借助化学小分子从功能角度直接切入蛋白质组的研究,能够直接对蛋白质组中感兴趣的靶酶蛋白的活性进行检测,为药物的发现提供强有力的支持.因此,ABPP技术被认为是基于功能的新一代蛋白质组学技术.随着ABPP分析技术和方法的不断成熟,其应用领域也不断扩展.最近一系列研究表明,今后ABPP分析技术可能成为病毒学研究的又一重要武器.本文综述了ABPP分析技术的基本原理及其在病毒学研究中的应用.
基金supported by the CAMS Innovation Fund for Medical Sciences(CIFMS,Nos.2021-I2M-1-069,2022-12M-2-002)Beijing Municipal Natural Science Foundation,China(No.7222259)。
文摘Schisandrin A is a natural dibenzocyclooctene lignan with potent neuroprotective activity.However,the specific mechanisms or direct target proteins have not been clarified up to now.In this study,we designed and synthesized the probes of schisandrin A with photoreactive diazirine and clickable alkyne to identify its direct target in SH-SY5Y cells by employing activity-based protein profiling(ABPP)technique.Ykt6 was prominent among the 13 proteins obtained with high confidence and we confirmed Ykt6 as the direct target of schisandrin A by CETSA,IF,SPR and knockdown assay.Functionally,schisandrin A protected the cells against the injury induced by glutamate by regulating autophagy via Ykt6.This discovery may provide a novel therapeutic option for various neuronal cell damage-mediated diseases.
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.
基金supported by the National Key Research and Development Program of China (2020YFA0908000)the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (ZYYCXTD-C-202002,China)+1 种基金the National Natural Science Foundation of China(81903588,81803456,82074098 and 81841001,China)the Fundamental Research Funds for the Central Public Welfare Research Institutes (ZXKT18003 and ZZ15-YQ-063,China)。
文摘Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron-and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species(ROS) and inducing ferroptosis in activated hepatic stellate cells(HSCs). By using activity-based protein profiling(ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay(CETSA), we show that celastrol directly binds to peroxiredoxins(PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6,through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1(HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2,PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis.
基金supported by the CAMS Innovation Fund for Medical Sciences(nos.2020-I2M-2-010 and 2016-I2M-1-011)the Drug Innovation Major Project(no.2018ZX09711-001)the National Natural Science Foundation of China(no.81974494).
文摘Seven photoaffinity-based and sixteen biotin-based berberine(BBR)probes were constructed and screened for their effects on c-Jun N-terminal protein kinases(JNK)phosphorylation(p-JNK)suppression at the cellular level.Taking active-photoaffinity probe 7c as a chemical tool,we first identified mitogen-activated protein kinase 7(MAP2K7),an upstream protein on the JNK/stress activated protein kinase(SAPK)pathway,as a direct proteomic target of BBR using activity-based protein profiling(ABPP)and other chemical proteomic techniques.Furthermore,BBR’s inhibitory effect on p-JNK was significantly attenuated in both the MAP2K7-knockdown and models,indicating a MAP2K7-dependent inhibition on the JNK signaling pathway.For the first time,we demonstrate the unique mechanism of BBR that directly targets MAP2K7 to inhibit p-JNK rather than JNK activity with the advantages of multiple activities and a good safety profile.
