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
Active endogenous metabolites regulate the viability of cells. This process is controlled by a series ofinteractions between small metabolites and large proteins. Previously, several studies had reported thatmetabolit...Active endogenous metabolites regulate the viability of cells. This process is controlled by a series ofinteractions between small metabolites and large proteins. Previously, several studies had reported thatmetabolite regulates the protein functions, such as diacylglycerol to protein kinase C, lactose regulationof the lac repressor, and HIF-1α stabilization by 2-hydroxyglutarate. However, decades old traditionalbiochemical methods are insufficient to systematically investigate the bio-molecular reactions for a high-throughput discovery. Here, we have reviewed an update on the recently developed chemical proteomicscalled activity-based protein profiling （ABPP）. ABPP is able to identify proteins interacted eithercovalently or non-covalently with metabolites significantly. Thus, ABPP will facilitate the characteriza-tion of specific metabolite regulating; proteins in human disease progression.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
Objective:Celastrol is a pentacyclic triterpenoid extracted from the traditional Chinese medicinal herb,Tripterygium wilfordii.This study aims to provide a scientific basis for the rational development and use of cela...Objective:Celastrol is a pentacyclic triterpenoid extracted from the traditional Chinese medicinal herb,Tripterygium wilfordii.This study aims to provide a scientific basis for the rational development and use of celastrol in breast cancer.Method:A quantitative chemical biology approach was used to investigate the protein targets and molecular mechanisms of celastrol in breast cancer cells.Results:Low-concentration celastrol exerted an anti-tumor effect by directly binding to hydroxysteroid dehydrogenase-like 2(HSDL2)and inhibiting its expression.Moreover,the expression of the pro-apoptotic protein,Bcl-2-associated X(BaX),increased,the level of the anti-apoptotic protein,B-cell lymphoma-2(Bcl-2),decreased,and the rate of apoptosis increased.After the transfection of cells with si-HSDL2,the apoptosis rate was similar to that observed after the administration of celastrol.However,apoptosis was reversed by the overexpression of HSDL2.Furthermore,our mass spectrometry(MS)data indicated a relationship between HSDL2 and the mitogen-activated protein kinase(MAPK)signaling pathway.We also found that the expression of HSDL2 was directly related to the degree of extracellular signal-regulated kinase(ERK)phosphorylation.Conclusion:Celastrol may promote apoptosis by suppressing the HSDL2/MAPK/ERK signaling pathway.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
Most drugs exert pharmacological effects through interaction with their target proteins.Therefore,drug target identification is a crucial step towards the understanding of the mechanism of drug action.It is also imper...Most drugs exert pharmacological effects through interaction with their target proteins.Therefore,drug target identification is a crucial step towards the understanding of the mechanism of drug action.It is also imperative to study the pharmacodynamics of a known drug,with an aim to discover the potentially unrevealed actions and thus refine its future clinical applications.Currently,drug-target identification is either through in vitro affinity chromatography-based approaches or in vivo activity-based protein profiling(ABPP)approaches.However,these approaches generally face difficulties discriminating specific drug targets from non-specific ones.To address this issue,we have come up with a strategy by coupling iTRAQTM(isobaric tags for relative and absolute quantitation)quantitative proteomics approach with clickable ABPP,to specifically and compre hensively identify drug targets in live cells.Using this approach,we identified the protein targets of andrographolide,a natural product with known anti-inflammation and anti-cancer effects,in live cancer cells.The identified target list not only confirmed the known functions of the drug but also revealed its potential novel application as a tumor metastasis inhibitor.We have also used this strategy,combining with a cleavable probe to identify the protein targets of aspirin and its binding sites.Our results revealed the roles of aspirin ininhibition of protein synthesis and induction of autophagy,which have been functionally validated.Our strategy is widely applicable to the identification of protein targets of covalent drugs.展开更多
Monoamine oxidase A(MAO-A)plays a critical role in the development of glioma and other neurological disorders.Specific analysis of MAO-A activities and its drug interactions in intact tissue is important for biologica...Monoamine oxidase A(MAO-A)plays a critical role in the development of glioma and other neurological disorders.Specific analysis of MAO-A activities and its drug interactions in intact tissue is important for biological and pharmacological research,but highly challenging with current chemical tools.Fluorogenic-inhibitor-based probes offer improved selectivity,sensitivity,and effectiveness to image and profile endogenous targets in an activity-based manner from mammalian cells,which are however rare.Herein,we report HD1 as the first fluorogenic-inhibitor-based probe that can selectively label endogenous MAO-A from various mammalian cells and clinical tissues.The probe was delicately designed based on N-propargyl tetrahydropyridine,a small MAO-A-specific fluorogenic and inhibitory war-head,so that the probe becomes fluorescent upon in situ enzymatic oxidation and covalent labeling of MAO-A.With the excellent binding affinity(in vitro K_(i)=285 n M)and fluorogenic properties,HD1 offers a promising approach to simultaneously image endogenous MAO-A activities by super-resolution fluorescence microscopy and study its drug interactions by subsequent activity-based protein profiling,in both live cells and human glioma tissues.展开更多
Conjunctival melanoma(CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B(CuB). We found that CuB remarkably in...Conjunctival melanoma(CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B(CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16,CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a Kdvalue of0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78-FOXM1-KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.展开更多
A resurging interest in targeted covalent inhibitors(TCIs)focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target.p97 is an emerging protein target for cancer therapy,vi...A resurging interest in targeted covalent inhibitors(TCIs)focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target.p97 is an emerging protein target for cancer therapy,viral infections and neurodegenerative diseases.Extensive efforts were devoted to the development of p97 inhibitors.The most promising inhibitor of p97 was in phase 1 clinical trials,but failed due to the off-target-induced toxicity,suggesting the selective inhibitors of p97 are highly needed.We report herein a new type of TCIs(i.e.,FL-18)that showed proteome-wide selectivity towards p97.Equipped with a Michael acceptor and a basic imidazole,FL-18 showed potent inhibition towards U87 MG tumor cells,and in proteome-wide profiling,selectively modified endogenous p97 as confirmed by in situ fluorescence scanning,label-free quantitative proteomics and functional validations.FL-18 selectively modified cysteine residues located within the D2 ATP site of p97.This covalent labeling of cysteine residue in p97 was verified by LC-MS/MS-based site-mapping and site-directed mutagenesis.Further structure-activity relationship(SAR)studies with FL-18 analogs were established.Collectively,FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity,thus providing a promising scaffold for cancer therapy.展开更多
Functional proteomics is a powerful approach to globally investigate protein functions and their biological significance.Activity-based protein profiling(ABPP)has been widely applied to explore the activity and ligand...Functional proteomics is a powerful approach to globally investigate protein functions and their biological significance.Activity-based protein profiling(ABPP)has been widely applied to explore the activity and ligandability of proteins in complex biological systems.Herein,we summarized our efforts in developing chemical probes and chemical proteomic pipelines to identify protein targets of certain post-translational modifications(PTMs),covalent drugs,natural products and cellular metabolites.Furthermore,computer-aided proteomic strategies were developed to globally identify functional residues including hyper-reactive cysteines,active sites of selenoproteins and proximal anchors for protein activation.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.
基金supported by the National Natural Science Foundation of China(No.81672440)Innovation Program of Science and Research from the DICP,CAS(No.DICP TMSR201601)the 100 Talents Program of Chinese Academy of Sciences
文摘Active endogenous metabolites regulate the viability of cells. This process is controlled by a series ofinteractions between small metabolites and large proteins. Previously, several studies had reported thatmetabolite regulates the protein functions, such as diacylglycerol to protein kinase C, lactose regulationof the lac repressor, and HIF-1α stabilization by 2-hydroxyglutarate. However, decades old traditionalbiochemical methods are insufficient to systematically investigate the bio-molecular reactions for a high-throughput discovery. Here, we have reviewed an update on the recently developed chemical proteomicscalled activity-based protein profiling （ABPP）. ABPP is able to identify proteins interacted eithercovalently or non-covalently with metabolites significantly. Thus, ABPP will facilitate the characteriza-tion of specific metabolite regulating; proteins in human disease progression.
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the National Key Research and Development Program of China(2020YFA0908000,2022YFC2303600)the National Natural Science Foundation of China(81903866,82274182)and the Central Public Welfare Research Institutes(ZZ13-YQ-105,ZZ15-YQ-065,ZZ14-YQ-058).
文摘Objective:Celastrol is a pentacyclic triterpenoid extracted from the traditional Chinese medicinal herb,Tripterygium wilfordii.This study aims to provide a scientific basis for the rational development and use of celastrol in breast cancer.Method:A quantitative chemical biology approach was used to investigate the protein targets and molecular mechanisms of celastrol in breast cancer cells.Results:Low-concentration celastrol exerted an anti-tumor effect by directly binding to hydroxysteroid dehydrogenase-like 2(HSDL2)and inhibiting its expression.Moreover,the expression of the pro-apoptotic protein,Bcl-2-associated X(BaX),increased,the level of the anti-apoptotic protein,B-cell lymphoma-2(Bcl-2),decreased,and the rate of apoptosis increased.After the transfection of cells with si-HSDL2,the apoptosis rate was similar to that observed after the administration of celastrol.However,apoptosis was reversed by the overexpression of HSDL2.Furthermore,our mass spectrometry(MS)data indicated a relationship between HSDL2 and the mitogen-activated protein kinase(MAPK)signaling pathway.We also found that the expression of HSDL2 was directly related to the degree of extracellular signal-regulated kinase(ERK)phosphorylation.Conclusion:Celastrol may promote apoptosis by suppressing the HSDL2/MAPK/ERK signaling pathway.
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.
文摘Most drugs exert pharmacological effects through interaction with their target proteins.Therefore,drug target identification is a crucial step towards the understanding of the mechanism of drug action.It is also imperative to study the pharmacodynamics of a known drug,with an aim to discover the potentially unrevealed actions and thus refine its future clinical applications.Currently,drug-target identification is either through in vitro affinity chromatography-based approaches or in vivo activity-based protein profiling(ABPP)approaches.However,these approaches generally face difficulties discriminating specific drug targets from non-specific ones.To address this issue,we have come up with a strategy by coupling iTRAQTM(isobaric tags for relative and absolute quantitation)quantitative proteomics approach with clickable ABPP,to specifically and compre hensively identify drug targets in live cells.Using this approach,we identified the protein targets of andrographolide,a natural product with known anti-inflammation and anti-cancer effects,in live cancer cells.The identified target list not only confirmed the known functions of the drug but also revealed its potential novel application as a tumor metastasis inhibitor.We have also used this strategy,combining with a cleavable probe to identify the protein targets of aspirin and its binding sites.Our results revealed the roles of aspirin ininhibition of protein synthesis and induction of autophagy,which have been functionally validated.Our strategy is widely applicable to the identification of protein targets of covalent drugs.
基金supported by the National Key R&D Program of China(2020YFA0709900)the National Natural Science Foundation of China(62288102,22077101,22004099)+3 种基金the Joint Research Funds of Department of Science&Technology of Shaanxi Province and Northwestern Polytechnical University(2020GXLH-Z-008,2020GXLH-Z-021,2020GXLH-Z-023)the Natural Science Foundation of Shaanxi Province(2022JM-130)the Key Research and Development Program of Shaanxi(2020ZDLGY13-04)the Fundamental Research Funds for the Central Universities
文摘Monoamine oxidase A(MAO-A)plays a critical role in the development of glioma and other neurological disorders.Specific analysis of MAO-A activities and its drug interactions in intact tissue is important for biological and pharmacological research,but highly challenging with current chemical tools.Fluorogenic-inhibitor-based probes offer improved selectivity,sensitivity,and effectiveness to image and profile endogenous targets in an activity-based manner from mammalian cells,which are however rare.Herein,we report HD1 as the first fluorogenic-inhibitor-based probe that can selectively label endogenous MAO-A from various mammalian cells and clinical tissues.The probe was delicately designed based on N-propargyl tetrahydropyridine,a small MAO-A-specific fluorogenic and inhibitory war-head,so that the probe becomes fluorescent upon in situ enzymatic oxidation and covalent labeling of MAO-A.With the excellent binding affinity(in vitro K_(i)=285 n M)and fluorogenic properties,HD1 offers a promising approach to simultaneously image endogenous MAO-A activities by super-resolution fluorescence microscopy and study its drug interactions by subsequent activity-based protein profiling,in both live cells and human glioma tissues.
基金supported by the National Mega-project for Innovative Drugs of China(2019ZX09721001-004-003)the National Natural Science Foundation of China(82003603 and 81872747)+1 种基金the Innovative Research Team of High-level Local Universities in Shanghai,the National Special Fund for State Key Laboratory of Bioreactor Engineering(2060204,China)Shanghai Frontiers Science Center of Optogenetic Techniques for Cell Metabolism(2021 Sci&Tech 03-28,China).
文摘Conjunctival melanoma(CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B(CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16,CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a Kdvalue of0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78-FOXM1-KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.
基金the funding support from Institute of Materia Medica,Peking Union Medical College,CAMS Innovation Fund for Medical Sciences(CIFMS)(2017-I2M-4-005,China)The Natural Science Foundation of China(No.22177136)+1 种基金the Synthetic Biology Research&Development Programme(SBP)of National Research Foundation(SBP-P4 and SBP-P8)of Singapore。
文摘A resurging interest in targeted covalent inhibitors(TCIs)focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target.p97 is an emerging protein target for cancer therapy,viral infections and neurodegenerative diseases.Extensive efforts were devoted to the development of p97 inhibitors.The most promising inhibitor of p97 was in phase 1 clinical trials,but failed due to the off-target-induced toxicity,suggesting the selective inhibitors of p97 are highly needed.We report herein a new type of TCIs(i.e.,FL-18)that showed proteome-wide selectivity towards p97.Equipped with a Michael acceptor and a basic imidazole,FL-18 showed potent inhibition towards U87 MG tumor cells,and in proteome-wide profiling,selectively modified endogenous p97 as confirmed by in situ fluorescence scanning,label-free quantitative proteomics and functional validations.FL-18 selectively modified cysteine residues located within the D2 ATP site of p97.This covalent labeling of cysteine residue in p97 was verified by LC-MS/MS-based site-mapping and site-directed mutagenesis.Further structure-activity relationship(SAR)studies with FL-18 analogs were established.Collectively,FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity,thus providing a promising scaffold for cancer therapy.
基金The authors acknowledge the supports by the National Natu-ral Science Foundation of China(Nos.21925701,91953109 and 21778004).
文摘Functional proteomics is a powerful approach to globally investigate protein functions and their biological significance.Activity-based protein profiling(ABPP)has been widely applied to explore the activity and ligandability of proteins in complex biological systems.Herein,we summarized our efforts in developing chemical probes and chemical proteomic pipelines to identify protein targets of certain post-translational modifications(PTMs),covalent drugs,natural products and cellular metabolites.Furthermore,computer-aided proteomic strategies were developed to globally identify functional residues including hyper-reactive cysteines,active sites of selenoproteins and proximal anchors for protein activation.
