RESUMO
Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.
Assuntos
Fumaratos/metabolismo , Leiomiomatose/metabolismo , Síndromes Neoplásicas Hereditárias/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Uterinas/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Cisteína , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Leiomiomatose/genética , Modelos Biológicos , Síndromes Neoplásicas Hereditárias/genética , Proteômica , Transdução de Sinais , Neoplasias Cutâneas/genética , Espectrometria de Massas em Tandem/métodos , Neoplasias Uterinas/genéticaRESUMO
Dysregulated metabolism can fuel cancer by altering the production of bioenergetic building blocks and directly stimulating oncogenic gene-expression programs. However, relatively few optical methods for the direct study of metabolites in cells exist. To address this need and facilitate new approaches to cancer treatment and diagnosis, herein we report an optimized chemical approach to detect the oncometabolite fumarate. Our strategy employs diaryl tetrazoles as cell-permeable photoinducible precursors to nitrileimines. Uncaging these species in cells and cell extracts enables them to undergo 1,3-dipolar cycloadditions with endogenous dipolarophile metabolites such as fumarate to form pyrazoline cycloadducts that can be readily detected by their intrinsic fluorescence. The ability to photolytically uncage diaryl tetrazoles provides greatly improved sensitivity relative to previous methods, and enables the facile detection of dysregulated fumarate metabolism through biochemical activity assays, intracellular imaging, and flow cytometry. Our studies showcase an intersection of bioorthogonal chemistry and metabolite reactivity that can be applied for biological profiling, imaging, and diagnostics.
Assuntos
Fluorescência , Fumaratos/análise , Fumaratos/efeitos da radiação , Linhagem Celular , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/efeitos da radiação , Fumaratos/metabolismo , Humanos , Microscopia Confocal , Estrutura Molecular , Imagem Óptica , Tetrazóis/químicaRESUMO
Surgical methods guided by exogenous fluorescent markers have the potential to define tissue types in real time. Small molecule dyes with efficient and selective renal clearance could enable visualization of the ureter during surgical procedures involving the abdomen and pelvis. These studies report the design and synthesis of a water soluble, net neutral C4'-O-alkyl heptamethine cyanine, Ureter-Label (UL)-766, with excellent properties for ureter visualization. This compound is accessed through a concise synthetic sequence involving an N- to O-transposition reaction that provides other inaccessible C4'-O-alkyl heptamethine cyanines. Unlike molecules containing a C4'-O-aryl substituent, which have also been used for ureter visualization, UL-766 is not reactive towards glutathione and the cellular proteome. In addition, rat models of abdominal surgery reveal that UL-766 undergoes efficient and nearly exclusive renal clearance in vivo. In total, this molecule represents a promising candidate for visualizing the ureter during a variety of surgical interventions.
Assuntos
Corantes Fluorescentes/química , Ureter/química , Animais , Biomarcadores/química , Relação Dose-Resposta a Droga , Corantes Fluorescentes/administração & dosagem , Corantes Fluorescentes/farmacocinética , Células HEK293 , Humanos , Injeções Intravenosas , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
Dysregulated metabolism is a hallmark of many diseases, including cancer. Methods to fluorescently detect metabolites have the potential to enable new approaches to cancer detection and imaging. However, fluorescent sensing methods for naturally occurring cellular metabolites are relatively unexplored. Here we report the development of a chemical approach to detect the oncometabolite fumarate. Our strategy exploits a known bioorthogonal reaction, the 1,3-dipolar cycloaddition of nitrileimines and electron-poor olefins, to detect fumarate via fluorescent pyrazoline cycloadduct formation. We demonstrate hydrazonyl chlorides serve as readily accessible nitrileimine precursors, whose reactivity and spectral properties can be tuned to enable detection of fumarate and other dipolarophile metabolites. Finally, we show this reaction can be used to detect enzyme activity changes caused by mutations in fumarate hydratase, which underlie the familial cancer predisposition syndrome hereditary leiomyomatosis and renal cell cancer. Our studies define a novel intersection of bioorthogonal chemistry and metabolite reactivity that may be harnessed to enable biological profiling, imaging, and diagnostic applications.
Assuntos
Alcenos/metabolismo , Carcinoma de Células Renais/metabolismo , Fumarato Hidratase/metabolismo , Fumaratos/metabolismo , Iminas/metabolismo , Neoplasias Renais/metabolismo , Alcenos/química , Carcinoma de Células Renais/patologia , Fumaratos/análise , Humanos , Iminas/química , Neoplasias Renais/patologia , Estrutura MolecularRESUMO
Lysine acetyltransferases (KATs) are key mediators of cell signaling. Methods capable of providing new insights into their regulation thus constitute an important goal. Here we report an optimized platform for profiling KAT-ligand interactions in complex proteomes using inhibitor-functionalized capture resins. This approach greatly expands the scope of KATs, KAT complexes, and CoA-dependent enzymes accessible to chemoproteomic methods. This enhanced profiling platform is then applied in the most comprehensive analysis to date of KAT inhibition by the feedback metabolite CoA. Our studies reveal that members of the KAT superfamily possess a spectrum of sensitivity to CoA and highlight NAT10 as a novel KAT that may be susceptible to metabolic feedback inhibition. This platform provides a powerful tool to define the potency and selectivity of reversible stimuli, such as small molecules and metabolites, that regulate KAT-dependent signaling.
Assuntos
Lisina Acetiltransferases/metabolismo , Catálise , Cromatografia Líquida , Coenzima A/metabolismo , Células HeLa , Humanos , Transdução de Sinais , Espectrometria de Massas em TandemRESUMO
Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls. Novel peptide-based probes for protein tyrosine phosphatases are needed to replace phosphotyrosine peptides and accelerate the field of tyrosine phosphatase substrate profiling. Here we review a type of peptidic probe for tyrosine phosphatases, which is based on the incorporation of the phosphotyrosine-mimic phosphocoumaryl amino propionic acid (pCAP) into peptides. The resulting fluorogenic pCAP peptides are dephosphorylated by tyrosine phosphatases with similar efficiency as the homologous phosphotyrosine peptides. pCAP peptides outperform phosphotyrosine peptides, providing an assay that is as robust, sensitive and facile as the non-peptidic fluorogenic probes on the market. Finally the use of pCAP can expand the range of phosphatase assays, facilitating the investigation of multiphosphorylated peptides and providing an in-gel assay for phosphatase activity.
Assuntos
Alanina/análogos & derivados , Bioensaio/métodos , Cumarínicos/química , Corantes Fluorescentes/química , Organofosfatos/química , Peptídeos/química , Proteínas Tirosina Fosfatases/química , Alanina/química , Eletroforese em Gel de Poliacrilamida , Humanos , Peptídeos/genética , Proteínas Tirosina Fosfatases/genética , Especificidade por SubstratoRESUMO
Assay design is an important variable that influences the outcome of an inhibitor screen. Here, we have investigated the hypothesis that protein tyrosine phosphatase inhibitors with improved biological activity could be identified from a screen by using a biologically relevant peptide substrate, rather than traditional phosphotyrosine mimetic substrates. A 2000-member library of drugs and drug-like compounds was screened for inhibitors of lymphoid tyrosine phosphatase (LYP) by using both a peptide substrate (Ac-ARLIEDNE-pCAP-TAREG-NH2, peptide 1) and a small-molecule phosphotyrosine mimetic substrate (difluoromethyl umbelliferyl phosphate, DiFMUP). The results demonstrate that compounds that inhibited enzyme activity on the peptide substrate had greater biological activity than compounds that only inhibited enzyme activity on DiFMUP. Finally, epigallocatechin-3,5-digallate was identified as the most potent inhibitor of lymphoid tyrosine phosphatase activity to date, with an IC50 of 50 nM and significant activity in T-cells. Molecular docking simulations provided a first model for binding of this potent inhibitor to LYP; this will constitute the platform for ongoing lead optimization efforts.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Peptídeos/farmacologia , Fosfotirosina/farmacologia , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Células Cultivadas , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Células Jurkat , Modelos Moleculares , Estrutura Molecular , Peptídeos/análise , Peptídeos/química , Fosfotirosina/análogos & derivados , Fosfotirosina/química , Proteínas Tirosina Fosfatases/metabolismo , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Statins are a class of drug widely prescribed for the prevention of cardiovascular disease, with pleiotropic cellular effects. Statins inhibit HMG-CoA reductase (HMGCR), which converts the metabolite HMG-CoA into mevalonate. Recent discoveries have shown HMG-CoA is a reactive metabolite that can non-enzymatically modify proteins and impact their activity. Therefore, we predicted that inhibition of HMGCR by statins might increase HMG-CoA levels and protein modifications. Upon statin treatment, we observe a strong increase in HMG-CoA levels and modification of only a single protein. Mass spectrometry identifies this protein as fatty acid synthase (FAS), which is modified on active site residues and, importantly, on non-lysine side-chains. The dynamic modifications occur only on a sub-pool of FAS that is located near HMGCR and alters cellular signaling around the ER and Golgi. These results uncover communication between cholesterol and lipid biosynthesis by the substrate of one pathway inhibiting another in a rapid and reversible manner.
Assuntos
Doenças Cardiovasculares , Inibidores de Hidroximetilglutaril-CoA Redutases , Doenças Cardiovasculares/prevenção & controle , Colesterol/metabolismo , Ácido Graxo Sintases , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Ácido Mevalônico/metabolismoRESUMO
Altered metabolite levels can drive epigenetic changes critical to development and disease. However, in many cases the specific protein-metabolite interactions that underlie this process remain enigmatic. In this review, we make the case that this fundamental missing information may be discovered by applying the tools of modern drug target validation to study endogenous metabolite pharmacology. We detail examples in which chemical proteomics has been applied to gain new insights into reversible and covalent metabolite signaling mechanisms, using acetyl-CoA and fumarate as case studies. Finally, we provide a brief survey of nascent chemical biology methods whose application to the study of endogenous metabolite pharmacology may further advance the field.
Assuntos
Epigênese Genética , Preparações Farmacêuticas/metabolismo , Acetilcoenzima A/metabolismo , Fumaratos/metabolismo , Humanos , Proteômica , Transdução de SinaisRESUMO
Short chain fatty acids (SCFAs) play a central role in health and disease. One function of these signaling molecules is to serve as precursors for short chain fatty acylation, a class of metabolically-derived posttranslational modifications (PTMs) that are established by lysine acetyltransferases (KATs) and lysine deacetylases (KDACs). Via this mechanism, short chain fatty acylation serves as an integrated reporter of metabolism as well as KAT and KDAC activity, and has the potential to illuminate the role of these processes in disease. However, few methods to study short chain fatty acylation exist. Here we report a bioorthogonal pro-metabolite strategy for profiling short chain fatty acylation in living cells. Inspired by the dietary component tributyrin, we synthesized a panel of ester-caged bioorthogonal short chain fatty acids. Cellular evaluation of these agents led to the discovery of an azido-ester that is metabolized to its cognate acyl-coenzyme A (CoA) and affords robust protein labeling profiles. We comprehensively characterize the metabolic dependence, toxicity, and histone deacetylase (HDAC) inhibitor sensitivity of these bioorthogonal pro-metabolites, and apply an optimized probe to identify novel candidate protein targets of short chain fatty acids in cells. Our studies showcase the utility of bioorthogonal pro-metabolites for unbiased profiling of cellular protein acylation, and suggest new approaches for studying the signaling functions of SCFAs in differentiation and disease.
RESUMO
The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASNKD) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASNKD elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASNKD ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade.
Assuntos
Ácido Graxo Sintase Tipo I/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Malonil Coenzima A/metabolismo , Neovascularização Retiniana/patologia , Serina-Treonina Quinases TOR/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ácido Graxo Sintase Tipo I/antagonistas & inibidores , Ácido Graxo Sintase Tipo I/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Orlistate/uso terapêutico , Processamento de Proteína Pós-Traducional , Neovascularização Retiniana/tratamento farmacológicoRESUMO
Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.
Assuntos
Ésteres/metabolismo , Compostos de Sulfidrila/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Acilação , Ésteres/química , Humanos , Modelos Moleculares , Estrutura Molecular , Proteômica , Compostos de Sulfidrila/química , Células Tumorais CultivadasRESUMO
Cell-permeating esters of 2-ketoglutarate (2-KG) have been synthesized through a convergent sequence from two modules in two and three steps, respectively. This route provides access to a full series of mono- and disubstituted 2-KG esters, enabling us to define the effect of regioisomeric masking on metabolite release and antihypoxic activity in cell-based assays. In addition to providing insight into the biological activity of cell permeable 2-KG esters, the straightforward and modular nature of this synthetic route may prove useful for the development of next-generation 2-KG analogues for diagnostic and therapeutic applications.
Assuntos
Ácidos Cetoglutáricos/síntese química , Fenômenos Biológicos , Permeabilidade da Membrana Celular , Ésteres , Ácidos Cetoglutáricos/química , Estrutura Molecular , EstereoisomerismoRESUMO
Somatic mutations in non-Hodgkin's lymphoma frequently activate EZH2, a protein methyltransferase responsible for H3K27 trimethylation. In this issue of Chemistry and Biology, Bradley and coworkers describe a new set of EZH2 inhibitors amenable to probing the targetable role of H3K27 trimethylation in lymphoma.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Histonas/metabolismo , Complexo Repressor Polycomb 2/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/toxicidade , Animais , HumanosRESUMO
We screened a small library of thiuram disulfides for inhibition of lymphoid tyrosine phosphatase (LYP) activity. The parent thiuram disulfide, disulfiram, inhibited LYP activity in vitro and in Jurkat Tâ cells, whereas diethyldithiocarbamate failed to inhibit LYP at the concentrations tested. Compound 13, an N-(2-thioxothiazolidin-4-one) analogue, was found to be the most potent LYP inhibitor in this series, with an IC50 value of 3â µM. Compound 13 inhibits LYP pseudo-irreversibly, as evidenced by the time-dependence of inhibition, with a K(i) value of 1.1â µM and a k(inact) value of 0.004â s⻹. The inhibition of LYP by compound 13 could not be reversed significantly by incubation with glutathione or by prolonged dialysis, but could be partially reversed by incubation with dithiothreitol. Compound 13 also inhibited LYP activity in Jurkat Tâ cells.
Assuntos
Dissulfetos/química , Inibidores Enzimáticos/química , Proteína Tirosina Fosfatase não Receptora Tipo 22/antagonistas & inibidores , Tiazóis/química , Tiram/química , Dissulfetos/metabolismo , Inibidores Enzimáticos/metabolismo , Humanos , Células Jurkat , Cinética , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 22/metabolismo , Relação Quantitativa Estrutura-Atividade , Tiazóis/metabolismo , Tiram/metabolismoRESUMO
Therapeutic inhibition of protein tyrosine phosphatase activity is a compelling yet challenging approach to the treatment of human disease. Toward this end, a library of 40 gold complexes with the general formula R(3)P-Au-Cl was screened to identify novel inhibitors of PTP activity. The most promising inhibitor obtained for the lymphoid tyrosine phosphatase LYP, (2-pyridine)(Ph(2))P-Au-Cl, is one of the most potent and selective LYP inhibitors identified to date with an IC(50) of 1.5 +/- 0.3 microM, 10-fold selectivity for LYP over PTP-PEST, HePTP, and CD45 in vitro, and activity in cellular studies as well.