RESUMO
UNLABELLED: Conventional wisdom ascribes metabolic reprogramming in cancer to meeting increased demands for intermediates to support rapid proliferation. Prior models have proposed benefits toward cell survival, immortality, and stress resistance, although the recent discovery of oncometabolites has shifted attention to chromatin targets affecting gene expression. To explore further effects of cancer metabolism and epigenetic deregulation, DNA repair kinetics were examined in cells treated with metabolic intermediates, oncometabolites, and/or metabolic inhibitors by tracking resolution of double-strand breaks (DSB) in irradiated MCF7 breast cancer cells. Disrupting cancer metabolism revealed roles for both glycolysis and glutaminolysis in promoting DSB repair and preventing accelerated senescence after irradiation. Targeting pathways common to glycolysis and glutaminolysis uncovered opposing effects of the hexosamine biosynthetic pathway (HBP) and tricarboxylic acid (TCA) cycle. Treating cells with the HBP metabolite N-acetylglucosamine (GlcNAc) or augmenting protein O-GlcNAcylation with small molecules or RNAi targeting O-GlcNAcase each enhanced DSB repair, while targeting O-GlcNAc transferase reversed GlcNAc's effects. Opposing the HBP, TCA metabolites including α-ketoglutarate blocked DSB resolution. Strikingly, DNA repair could be restored by the oncometabolite 2-hydroxyglutarate (2-HG). Targeting downstream effectors of histone methylation and demethylation implicated the PRC1/2 polycomb complexes as the ultimate targets for metabolic regulation, reflecting known roles for Polycomb group proteins in nonhomologous end-joining DSB repair. Our findings that epigenetic effects of cancer metabolic reprogramming may promote DNA repair provide a molecular mechanism by which deregulation of metabolism may not only support cell growth but also maintain cell immortality, drive therapeutic resistance, and promote genomic instability. IMPLICATIONS: By defining a pathway from deregulated metabolism to enhanced DNA damage response in cancer, these data provide a rationale for targeting downstream epigenetic effects of metabolic reprogramming to block cancer cell immortality and overcome resistance to genotoxic stress.
Assuntos
Reparo do DNA , Epigênese Genética , Glutamina/metabolismo , Glicólise , Neoplasias/metabolismo , Acetilglucosamina/farmacologia , Senescência Celular , DNA/efeitos da radiação , Instabilidade Genômica , Glicólise/efeitos dos fármacos , Humanos , Células MCF-7 , Neoplasias/genética , Interferência de RNARESUMO
Development of cell-permeable small molecules that target enzymes involved in energy metabolism remains important yet challenging. We describe here the discovery of a new class of compounds with a nutrient-dependent cytotoxicity profile that arises from pharmacological inhibition of fumarate hydratase (also known as fumarase). This finding was enabled by a high-throughput screen of a diverse chemical library in a panel of human cancer cell lines cultured under different growth conditions, followed by subsequent structure-activity optimization and target identification. While the highest cytotoxicity was observed under low glucose concentrations, the antiproliferative activities and inhibition of oxygen consumption rates in cells were distinctly different from those displayed by typical inhibitors of mitochondrial oxidative phosphorylation. The use of a photoaffinity labeling strategy identified fumarate hydratase as the principal pharmacological target. Final biochemical studies confirmed dose-dependent, competitive inhibition of this enzyme in vitro, which was fully consistent with the initially observed growth inhibitory activity. Our work demonstrates how the phenotypic observations combined with a successful target identification strategy can yield a useful class of pharmacological inhibitors of an enzyme involved in the operation of tricarboxylic acid cycle.
Assuntos
Inibidores Enzimáticos/farmacologia , Fumarato Hidratase/antagonistas & inibidores , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Metabolismo Energético/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Fumarato Hidratase/metabolismo , Glucose/metabolismo , Ensaios de Triagem em Larga Escala , HumanosRESUMO
Cycloaddition uncovered: The title reaction produces novel polycyclic compounds with high efficiency and excellent diastereoselectivity under mild reaction conditions. A small-molecule library, synthesized using this reaction, yielded a novel chemotype which inhibited glycolytic ATP production by blocking glucose uptake in CHO-K1 cells. DMF=N,N-dimethylformamide, Tf=trifluoromethanesulfonyl, TIPS=triisopropylsilyl.
Assuntos
Alcinos/química , Reação de Cicloadição/métodos , Catálise , Ciclização , Glicólise , Estrutura Molecular , EstereoisomerismoRESUMO
We have developed a synthetic strategy that mimics the diversity-generating power of monoterpenoid indole alkaloid biosynthesis. Our general approach goes beyond diversification of a single natural product-like substructure and enables production of a highly diverse collection of small molecules. The reaction sequence begins with rapid and highly modular assembly of the tetracyclic indoloquinolizidine core, which can be chemoselectively processed into several additional skeletally diverse structural frameworks. The general utility of this approach was demonstrated by parallel synthesis of two representative chemical libraries containing 847 compounds with favorable physicochemical properties to enable its subsequent broad pharmacological evaluation.
Assuntos
Alcaloides/síntese química , Monoterpenos/síntese química , Bibliotecas de Moléculas Pequenas/síntese química , Alcaloides/química , Modelos Moleculares , Estrutura Molecular , Monoterpenos/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The identification of new reactions expands our knowledge of chemical reactivity and enables new synthetic applications. Accelerating the pace of this discovery process remains challenging. We describe a highly effective and simple platform for screening a large number of potential chemical reactions in order to discover and optimize previously unknown catalytic transformations, thereby revealing new chemical reactivity. Our strategy is based on labelling one of the reactants with a polyaromatic chemical tag, which selectively undergoes a photoionization/desorption process upon laser irradiation, without the assistance of an external matrix, and enables rapid mass spectrometric detection of any products originating from such labelled reactants in complex reaction mixtures without any chromatographic separation. This method was successfully used for high-throughput discovery and subsequent optimization of two previously unknown benzannulation reactions.
Assuntos
Descoberta de Drogas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
We describe the development of efficient benzannulations of siloxy alkynes with pyridinium and isoquinolinium salts. Such reactions are successfully promoted by a stoichiometric amount of silver(I) benzolate under mild reaction conditions. This process proceeds via a formal inverse-electron demand Diels-Alder reaction, followed by fragmentation of the initially produced bicyclic adducts to deliver a range of synthetically useful phenols and naphthols.
RESUMO
We describe a unified synthetic strategy for efficient assembly of four new heterocyclic libraries. The synthesis began by creating a range of structurally diverse pyrrolidinones or piperidinones. Such compounds were obtained in a simple one-flask operation starting with readily available amines, ketoesters, and unsaturated anhydrides. The use of tetrahydropyran-containing ketoesters, which were rapidly assembled by our Prins cyclization protocol, enabled efficient fusion of pyran and piperidinone cores. A newly developed Au(I)-catalyzed cycloisomerization of alkyne-containing enamides further expanded heterocyclic diversity by providing rapid entry into a wide range of bicyclic and tricyclic dienamides. The final stage of the process entailed diversification of each of the initially produced carboxylic acids using a fully automated platform for amide synthesis, which delivered 1872 compounds in high diastereomeric and chemical purity.
Assuntos
Amidas/síntese química , Compostos Heterocíclicos/síntese química , Compostos Organoáuricos/química , Bibliotecas de Moléculas Pequenas/síntese química , Amidas/química , Catálise , Ciclização , Compostos Heterocíclicos/química , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/químicaRESUMO
A highly effective silver-catalyzed formal inverse electron-demand Diels-Alder reaction of 1,2-diazines and siloxy alkynes has been developed. The reactions provide ready access to a wide range of siloxy naphthalenes and anthracenes, which are formed in good to high yields, under mild reaction conditions, using low catalyst loadings.
Assuntos
Alcinos/química , Elétrons , Hidrazinas/química , Compostos de Organossilício/química , Compostos de Organossilício/síntese química , Prata/química , Catálise , Ciclização , Estrutura MolecularRESUMO
Multicomponent reactions are employed extensively in many areas of organic chemistry. Despite significant progress, the discovery of such enabling transformations remains challenging. Here, we present the development of a parallel, label-free reaction-discovery platform that can be used in the identification of new multicomponent transformations. Our approach is based on parallel mass spectrometric screening of interfacial chemical reactions on arrays of self-assembled monolayers. This strategy enabled the identification of a simple organic phosphine that can catalyse a previously unknown condensation of siloxyalkynes, aldehydes and amines to produce 3-hydroxyamides with high efficiency and diastereoselectivity. The reaction was further optimized using solution-phase methods.
Assuntos
Técnicas de Química Combinatória , Espectrometria de Massas , Aldeídos/química , Amidas/química , Aminas/química , Catálise , Ouro/química , Modelos Moleculares , Fosfinas/química , Siloxanas/química , Sulfetos/químicaRESUMO
Inhibition of bacterial transcription represents an effective and clinically validated anti-infective chemotherapeutic strategy. We describe the evolution of our approach to the streptolydigin class of antibiotics that target bacterial RNA polymerases (RNAPs). This effort resulted in the synthesis and biological evaluation of streptolydigin, streptolydiginone, streptolic acid, and a series of new streptolydigin-based agents. Subsequent biochemical evaluation of RNAP inhibition demonstrated that the presence of both streptolic acid and tetramic acid subunits was required for activity of this class of antibiotics. In addition, we identified 10,11-dihydrostreptolydigin as a new RNAP-targeting agent, which was assembled with high synthetic efficiency of 15 steps in the longest linear sequence. Dihydrostreptolydigin inhibited three representative bacterial RNAPs and displayed in vitro antibacterial activity against S. salivarius . The overall increase in synthetic efficiency combined with substantial antibacterial activity of this fully synthetic antibiotic demonstrates the power of organic synthesis in enabling design and comprehensive in vitro pharmacological evaluation of new chemical agents that target bacterial transcription.
Assuntos
Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Streptococcus/efeitos dos fármacos , Aminoglicosídeos/síntese química , Aminoglicosídeos/química , Antibacterianos/síntese química , Antibacterianos/química , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Thermus/químicaRESUMO
We have developed an efficient strategy to a skeletally diverse chemical library, which entailed a sequence of enyne cycloisomerization, [4 + 2] cycloaddition, alkene dihydroxylation, and diol carbamylation. Using this approach, only 16 readily available building blocks were needed to produce a representative 191-member library, which displayed broad distribution of molecular shapes and excellent physicochemical properties. This library further enabled identification of a small molecule, which effectively suppressed glycolytic production of ATP and lactate in CHO-K1 cell line, representing a potential lead for the development of a new class of glycolytic inhibitors.
Assuntos
Trifosfato de Adenosina/biossíntese , Glicólise/efeitos dos fármacos , Compostos Heterocíclicos com 3 Anéis , Animais , Células CHO , Cricetinae , Cricetulus , Glicólise/fisiologia , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/farmacologiaRESUMO
Oxidative phosphorylation (OXPHOS) and glycolysis are the two main pathways that control energy metabolism of a cell. The Warburg effect, in which glycolysis remains active even under aerobic conditions, is considered a key driver for cancer cell proliferation, malignancy, metastasis, and therapeutic resistance. To target aerobic glycolysis, we exploited the complementary roles of OXPHOS and glycolysis in ATP synthesis as the basis for a chemical genetic screen, enabling rapid identification of novel small-molecule inhibitors of facilitative glucose transport. Blocking mitochondrial electron transport with antimycin A or leucascandrolide A had little effect on highly glycolytic A549 lung carcinoma cells, but adding known glycolytic inhibitors 2-deoxy-D-glucose, iodoacetate or cytochalasin B, rapidly depleted intracellular ATP, displaying chemical synthetic lethality. Based on this principle, we exposed antimycin A-treated A549 cells to a newly synthesized 955 member diverse scaffold small-molecule library, screening for compounds that rapidly depleted ATP levels. Two compounds potently suppressed ATP synthesis, induced G1 cell-cycle arrest and inhibited lactate production. Pathway analysis revealed that these novel probes inhibited GLUT family of facilitative transmembrane transporters but, unlike cytochalasin B, had no effect on the actin cytoskeleton. Our work illustrated the utility of a pairwise chemical genetic screen for discovery of novel chemical probes, which would be useful not only to study the system-level organization of energy metabolism but could also facilitate development of drugs targeting upregulation of aerobic glycolysis in cancer.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Glucose/metabolismo , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Animais , Antimicina A/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Fase G1/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Glicólise/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ácido Láctico/biossíntese , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Fatores de TempoRESUMO
Streptolydigin is a highly potent, broad-spectrum antibiotic produced by Streptomyces lydicus, which inhibits bacterial RNA polymerase. We describe the first synthesis of streptolydigin, which was assembled in a highly convergent and fully stereocontrolled fashion with a longest linear sequence of 24 steps starting from commercially available precursors. The assembly process entailed preparation of fully elaborated streptolic and ydiginic subunits of the natural product, followed by a highly efficient union in a three-step one-pot procedure, which included Dieckmann cyclization with a concomitant imide opening, Horner-Wadsworth-Emmons olefination, and desilylation.
Assuntos
Aminoglicosídeos/síntese química , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Streptomyces/química , Aminoglicosídeos/químicaAssuntos
Estresse Oxidativo , Bibliotecas de Moléculas Pequenas/síntese química , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Ensaios de Triagem em Larga Escala , Humanos , Espécies Reativas de Oxigênio/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Compostos de Sulfonilureia/síntese química , Compostos de Sulfonilureia/química , Compostos de Sulfonilureia/farmacologiaRESUMO
We describe structure-based design and chemical synthesis of a simplified analog of bistramide A, which potently and reversibly binds monomeric actin with a K(d) of 9.0 nM, depolymerizes filamentous actin in vitro and in A549 (nonsmall cell lung cancer) cells, inhibits growth of cancer cell lines in vitro at submicromolar concentrations, and significantly suppresses proliferation of A549 cells in a nude mice tumor xenograft model in terms of both tumor growth delay and average tumor volume. This study provides a conceptual framework for the design and development of new antiproliferative compounds that target cytoskeletal organization of cancer cells in vivo by a combination of reversible G-actin binding and effective F-actin severing.
Assuntos
Acetamidas/química , Actinas/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias/metabolismo , Piranos/química , Animais , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Camundongos , Camundongos Nus , Compostos de Espiro/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We describe the assembly of a 960-member library of tricyclic 2,3-dihydro-4-quinolones using a combination of solution-phase high-throughput organic synthesis and parallel chromatographic purification. The library was produced with high efficiency and complete chemo- and diastereoselectivity by diversification of an azide-bearing quinolone via a sequence of [4 + 2] cycloadditions, N-acylations, and reductive aminations. The azide-functionalization of this library is designed to facilitate subsequent preparation of fluorescent or affinity probes, as well as small-molecule/surface conjugation.
Assuntos
4-Quinolonas/síntese química , Azidas/síntese química , 4-Quinolonas/química , Azidas/química , Técnicas de Química Combinatória/métodos , Ciclização , Espectroscopia de Ressonância Magnética , Modelos Químicos , Soluções/química , EstereoisomerismoRESUMO
We describe the development of a silver-catalyzed carbonyl olefination employing electron rich siloxy alkynes. This process constitutes an efficient synthesis of trisubstituted unsaturated esters, and represents an alternative to the widely utilized Horner-Wadsworth-Emmons reaction. Excellent diastereoselectivities are observed for a range of aldehydes using either 1-siloxy-1-propyne or 1-siloxy-1-hexyne. This mild catalytic process also enables chemoselective olefination of aldehydes in the presence of either ester or ketone functionality. Furthermore, since no by-products are generated, this catalytic process is perfectly suited for development of sequential reactions that can be carried out in a single flask.
RESUMO
Formins stimulate actin filament assembly for fundamental cellular processes including division, adhesion, establishing polarity, and motility. A formin inhibitor would be useful because most cells express multiple formins whose functions are not known and because metastatic tumor formation depends on the deregulation of formin-dependent processes. We identified a general small molecule inhibitor of formin homology 2 domains (SMIFH2) by screening compounds for the ability to prevent formin-mediated actin assembly in vitro. SMIFH2 targets formins from evolutionarily diverse organisms including yeast, nematode worm, and mice, with a half-maximal inhibitor concentration of approximately 5 to 15 microM. SMIFH2 prevents both formin nucleation and processive barbed end elongation and decreases formin's affinity for the barbed end. Furthermore, low micromolar concentrations of SMIFH2 disrupt formin-dependent, but not Arp2/3 complex-dependent, actin cytoskeletal structures in fission yeast and mammalian NIH 3T3 fibroblasts.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Actinas/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas dos Microfilamentos/antagonistas & inibidores , Tionas/farmacologia , Uracila/análogos & derivados , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/química , Animais , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Forminas , Humanos , Camundongos , Microscopia de Fluorescência , Células NIH 3T3 , Estrutura Terciária de Proteína , Pirimidinonas/química , Pirimidinonas/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Tionas/química , Tionas/toxicidade , Uracila/química , Uracila/farmacologia , Uracila/toxicidadeRESUMO
Leucascandrolide A and neopeltolide are structurally homologous marine natural products that elicit potent antiproliferative profiles in mammalian cells and yeast. The scarcity of naturally available material has been a significant barrier to their biochemical and pharmacological evaluation. We developed practical synthetic access to this class of natural products that enabled the determination of their mechanism of action. We demonstrated effective cellular growth inhibition in yeast, which was substantially enhanced by substituting glucose with galactose or glycerol. These results, along with genetic analysis of determinants of drug sensitivity, suggested that leucascandrolide A and neopeltolide may inhibit mitochondrial ATP synthesis. Evaluation of the activity of the four mitochondrial electron transport chain complexes in yeast and mammalian cells revealed cytochrome bc(1) complex as the principal cellular target. This result provided the molecular basis for the potent antiproliferative activity of this class of marine macrolides, thus identifying them as new biochemical tools for investigation of eukaryotic energy metabolism.
Assuntos
Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Macrolídeos , Sesquiterpenos , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Humanos , Macrolídeos/síntese química , Macrolídeos/química , Macrolídeos/farmacologia , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/enzimologia , Modelos Moleculares , Estrutura Molecular , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Sesquiterpenos/síntese química , Sesquiterpenos/química , Sesquiterpenos/farmacologiaRESUMO
This study provides comprehensive characterization of the mode of action of bistramide A and identifies structural requirements of bistramide-based compounds that are responsible for severing actin filaments and inhibiting growth of cancer cells in vitro and in vivo. We rationally designed and assembled a series of structural analogs of the natural product, including a fluorescently labeled conjugate. We used TIRF microscopy to directly observe actin filament severing by this series of small molecules, which established that the combination of the spiroketal and the amide subunits was sufficient to enable rapid actin filament disassembly in vitro. In addition, we demonstrated that the enone subunit of bistramide A is responsible for covalent modification of the protein in vitro and in A549 cells, resulting in further increase in the cytotoxicity of the natural product. Our results demonstrate that bistramide A elicits its potent antiproliferative activity by a dual mechanism of action, which entails both severing of actin filaments and covalent sequestration of monomeric actin in the cell.