RESUMO
BACKGROUND: Glioblastoma (GBM) is an aggressive brain cancer associated with poor prognosis, intrinsic heterogeneity, plasticity, and therapy resistance. In some GBMs, cell proliferation is fueled by a transcriptional regulator, repressor element-1 silencing transcription factor (REST). RESULTS: Using CRISPR/Cas9, we identified GBM cell lines dependent on REST activity. We developed new small molecule inhibitory compounds targeting small C-terminal domain phosphatase 1 (SCP1) to reduce REST protein level and transcriptional activity in glioblastoma cells. Top leads of the series like GR-28 exhibit potent cytotoxicity, reduce REST protein level, and suppress its transcriptional activity. Upon the loss of REST protein, GBM cells can potentially compensate by rewiring fatty acid metabolism, enabling continued proliferation. Combining REST inhibition with the blockade of this compensatory adaptation using long-chain acyl-CoA synthetase inhibitor Triacsin C demonstrated substantial synergetic potential without inducing hepatotoxicity. CONCLUSIONS: Our results highlight the efficacy and selectivity of targeting REST alone or in combination as a therapeutic strategy to combat high-REST GBM.
Assuntos
Glioblastoma , Fatores de Transcrição , Humanos , Glioblastoma/tratamento farmacológico , Regulação da Expressão Gênica , Encéfalo , AgressãoRESUMO
Working in tandem with kinases via a dynamic interplay of phosphorylation and dephosphorylation of proteins, phosphatases regulate many cellular processes and thus represent compelling therapeutic targets. Here we leverage ultraviolet photodissociation to shed light on the binding characteristics of two covalent phosphatase inhibitors, T65 and rabeprazole, and their respective interactions with the human small C-terminal domain phosphatase 1 (SCP1) and its single-point mutant C181A, in which a nonreactive alanine replaces one key reactive cysteine. Top-down MS/MS analysis is used to localize the binding of T65 and rabeprazole on the two proteins and estimate the relative reactivities of each cysteine residue.
Assuntos
Espectrometria de Massas em Tandem , Raios Ultravioleta , Humanos , Espectrometria de Massas em Tandem/métodos , Cisteína/química , Cisteína/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligação Proteica , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/metabolismo , Modelos MolecularesRESUMO
Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.
Assuntos
Amidas , Ácidos e Sais Biliares , Ésteres , Ácidos Graxos , Metabolômica , Animais , Humanos , Bifidobacterium/metabolismo , Ácidos e Sais Biliares/química , Ácidos e Sais Biliares/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Clostridium/metabolismo , Estudos de Coortes , Doença de Crohn/metabolismo , Enterococcus/metabolismo , Ésteres/química , Ésteres/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Metabolômica/métodos , Fenótipo , Receptor de Pregnano X/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Amidas/química , Amidas/metabolismoRESUMO
Eupalinilide E was assessed for ex-vivo expansion activity on hematopoietic stem cells (HSCs) from human cord blood (CB) CD34+ cells in serum-free, SCF, TPO and FL stimulated 7 day cultures. Eupalinilide E ex-vivo enhanced phenotyped (p) HSCs and glycolysis of CD34+ cells isolated 7 days after culture as measured by extracellular acidification rate, but did not alone show enhanced NSG engrafting capability of HSCs as determined by chimerism and numbers of SCID Repopulating cells, a quantitative measure of functional human HSCs. This is another example of pHSCs not necessarily recapitulating functional activity of these cells. Lack of effect on engrafting HSCs may be due to a number of possibilities, including down regulation of CXCR4 or of the homing capacity of these treated cells. However, Eupalinilide did act in an additive to synergistic fashion with UM171 to enhance ex vivo expansion of both pHSCs, and functionally engrafting HSCs. While reasons for the disconnect between pHSC and function of HSCs with Eupalinilide E alone cultured CB CD34+ cells is yet to be determined, the data suggest possible future use of Eupalinilide and UM171 together to enhance ex vivo production of CB HSCs for clinical hematopoietic cell transplantation.
Assuntos
Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Indóis/farmacologia , Pirimidinas/farmacologia , Sesquiterpenos/farmacologia , Animais , Antígenos CD34/análise , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/farmacologia , Sangue Fetal/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos SCIDRESUMO
Thiopeptides are a class of natural products with untapped therapeutic potential. To expand the methods available for the scaled production of these antibiotics, we report the laboratory synthesis of micrococcin P1 showcasing thiazole forming reactions of cysteine derivatives and nitriles followed by oxidation. In most instances, this thiazole forming sequence does not require chromatography and proved scalable. Using this approach, 199 mg of micrococcin P1 was generated in a single synthetic sequence.
Assuntos
Bacteriocinas/síntese química , Cisteína/análogos & derivados , Nitrilas/química , Tiazóis/síntese química , Cisteína/química , Tiazóis/químicaRESUMO
Dietary habits have been associated with alterations of the human gut resident microorganisms contributing to obesity, diabetes and cancer1. In Western diets, red meat is a frequently eaten food2, but long-term consumption has been associated with increased risk of disease3,4. Red meat is enriched in N-glycolylneuraminic acid (Neu5Gc) that cannot be synthesized by humans5. However, consumption can cause Neu5Gc incorporation into cell surface glycans6, especially in carcinomas4,7. As a consequence, an inflammatory response is triggered when Neu5Gc-containing glycans encounter circulating anti-Neu5Gc antibodies8,9. Although bacteria can use free sialic acids as a nutrient source10-12, it is currently unknown if gut microorganisms contribute to releasing Neu5Gc from food. We found that a Neu5Gc-rich diet induces changes in the gut microbiota, with Bacteroidales and Clostridiales responding the most. Genome assembling of mouse and human shotgun metagenomic sequencing identified bacterial sialidases with previously unobserved substrate preference for Neu5Gc-containing glycans. X-ray crystallography revealed key amino acids potentially contributing to substrate preference. Additionally, we verified that mouse and human sialidases were able to release Neu5Gc from red meat. The release of Neu5Gc from red meat using bacterial sialidases could reduce the risk of inflammatory diseases associated with red meat consumption, including colorectal cancer4 and atherosclerosis13.
Assuntos
Bactérias/enzimologia , Dieta , Microbioma Gastrointestinal , Ácidos Neuramínicos/metabolismo , Neuraminidase/genética , Polissacarídeos/metabolismo , Carne Vermelha/análise , Animais , Bactérias/classificação , Bacteroides/enzimologia , Bacteroides/genética , Clostridiales/enzimologia , Clostridiales/genética , Cristalografia por Raios X , Fezes/química , Fezes/microbiologia , Feminino , Humanos , Masculino , Metagenômica , Camundongos , Camundongos Endogâmicos C57BL , Neuraminidase/metabolismo , Polissacarídeos/químicaRESUMO
Palmitic acid esters of hydroxy stearic acids (PAHSAs) are bioactive lipids with antiinflammatory and antidiabetic effects. PAHSAs reduce ambient glycemia and improve glucose tolerance and insulin sensitivity in insulin-resistant aged chow- and high-fat diet-fed (HFD-fed) mice. Here, we aimed to determine the mechanisms by which PAHSAs improve insulin sensitivity. Both acute and chronic PAHSA treatment enhanced the action of insulin to suppress endogenous glucose production (EGP) in chow- and HFD-fed mice. Moreover, chronic PAHSA treatment augmented insulin-stimulated glucose uptake in glycolytic muscle and heart in HFD-fed mice. The mechanisms by which PAHSAs enhanced hepatic insulin sensitivity included direct and indirect actions involving intertissue communication between adipose tissue and liver. PAHSAs inhibited lipolysis directly in WAT explants and enhanced the action of insulin to suppress lipolysis during the clamp in vivo. Preventing the reduction of free fatty acids during the clamp with Intralipid infusion reduced PAHSAs' effects on EGP in HFD-fed mice but not in chow-fed mice. Direct hepatic actions of PAHSAs may also be important, as PAHSAs inhibited basal and glucagon-stimulated EGP directly in isolated hepatocytes through a cAMP-dependent pathway involving Gαi protein-coupled receptors. Thus, this study advances our understanding of PAHSA biology and the physiologic mechanisms by which PAHSAs exert beneficial metabolic effects.
Assuntos
Resistência à Insulina/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Estearatos/farmacologia , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , AMP Cíclico/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glucagon/farmacologia , Técnicas In Vitro , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estearatos/administração & dosagemRESUMO
Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids. Here we identify the linoleic acid ester of 13-hydroxy linoleic acid (13-LAHLA) as an anti-inflammatory lipid. An oat oil fraction and FAHFA-enriched extract from this fraction showed anti-inflammatory activity in a lipopolysaccharide-induced cytokine secretion assay. Structural studies identified three LAHLA isomers (15-, 13-, and 9-LAHLA) as being the most abundant FAHFAs in the oat oil fraction. Of these LAHLAs, 13-LAHLA is the most abundant LAHLA isomer in human serum after ingestion of liposomes made of fractionated oat oil, and it is also the most abundant endogenous LAHLA in mouse and human adipose tissue. As a result, we chemically synthesized 13-LAHLA for biological assays. 13-LAHLA suppresses lipopolysaccharide-stimulated secretion of cytokines and expression of pro-inflammatory genes. These studies identify LAHLAs as an evolutionarily conserved lipid with anti-inflammatory activity in mammalian cells.
Assuntos
Anti-Inflamatórios/química , Avena/química , Ésteres/química , Ácidos Linoleicos/química , Tecido Adiposo/química , Tecido Adiposo/metabolismo , Animais , Anti-Inflamatórios/análise , Anti-Inflamatórios/farmacologia , Avena/metabolismo , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Citocinas/metabolismo , Humanos , Lipopolissacarídeos/toxicidade , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Espectrometria de Massas , Camundongos , Óleos de Plantas/química , Óleos de Plantas/farmacologia , Células RAW 264.7 , EstereoisomerismoRESUMO
To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.
Assuntos
Antimaláricos/farmacologia , Quimioprevenção , Descoberta de Drogas , Malária/prevenção & controle , Plasmodium/efeitos dos fármacos , Antimaláricos/química , Antimaláricos/isolamento & purificação , Antimaláricos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Humanos , Mitocôndrias/efeitos dos fármacos , Plasmodium/crescimento & desenvolvimentoRESUMO
Following the discovery that the guaianolide natural product eupalinilide E promotes the expansion of hematopoietic stem and progenitor cells; the development of a synthetic route to provide laboratory access to the natural product became a priority. Exploration of multiple synthetic routes yielded an approach that has permitted a scalable synthesis of the natural product. Two routes that failed to access eupalinilide E were triaged either as a result of providing an incorrect diastereomer or due to lack of synthetic efficiency. The successful strategy relied on late-stage allylic oxidations at two separate positions of the molecule, which significantly increased the breadth of reactions that could be used to this point. Subsequent to C-H bond oxidation, adaptations of existing chemical transformations were required to permit chemoselective reduction and oxidation reactions. These transformations included a modified Luche reduction and a selective homoallylic alcohol epoxidation.
Assuntos
Sesquiterpenos/síntese química , Laboratórios , OxirreduçãoRESUMO
Improving the ex vivo and in vivo production of hematopoietic stem and progenitor cells (HSPCs) has the potential to address the short supply of these cells that are used in the treatment of various blood diseases and disorders. Eupalinilide E promotes the expansion of human HSPCs and inhibits subsequent differentiation, leading to increased numbers of clinically useful cells. This natural product represents an important tool to uncover new methods to drive expansion while inhibiting differentiation. However, in the process of examining these effects, which occur through a novel mechanism, the natural product was consumed, which limited additional investigation. To provide renewed and improved access to eupalinilide E, a laboratory synthesis has been developed and is reported herein. The synthetic route can access >400 mg in a single batch, employing reactions conducted on useful scales in a single vessel. Key transformations enabling the approach include a diastereoselective borylative enyne cyclization and a late-stage double allylic C-H oxidation as well as adapted Luche reduction and aluminum-mediated epoxidation reactions to maximize the synthetic efficiency. Retesting of the synthetic eupalinilide E confirmed the compound's ability to expand HSPCs and inhibit differentiation.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Sesquiterpenos/síntese química , Sesquiterpenos/farmacologia , Células-Tronco/efeitos dos fármacos , Alumínio/química , Antígenos CD34/biossíntese , Diferenciação Celular , Proliferação de Células , Células-Tronco Hematopoéticas/metabolismo , Humanos , Oxirredução , Células-Tronco/metabolismo , EstereoisomerismoRESUMO
A series of pentacyclic tritperpenes found in Perilla frutescens (P. frutescens), including ursolic acid (UA), oleanolic acid (OA), corosolic acid (CA), 3-epi-corosolic acid (3-epiCA), maslinic acid (MA), and 3-epi-maslinic acid (3-epiMA) were evaluated for their effects on epidermal cell signaling, proliferation, and skin inflammation in relation to their ability to inhibit skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) and compared to UA as the prototype compound. All compounds were given topically 30 min prior to each TPA application and significantly inhibited skin tumor promotion. 3-epiCA and MA were significantly more effective than UA at inhibiting tumor development. All of these compounds significantly inhibited epidermal proliferation induced by TPA, however, CA, 3-epiCA and MA were more effective than UA. All compounds also reduced skin inflammation (assessed by infiltration of mast cells and T-cells) and inflammatory gene expression induced by TPA, however, 3-epiCA and MA were again more effective than UA. The greater ability of 3-epiCA and MA to inhibit skin tumor promotion was associated with greater reduction of Cox-2 and Twist1 proteins and inhibition of activation (i.e., phosphorylation) of IGF-1R, STAT3 and Src. Further study of these compounds, especially 3-epiCA and MA, for chemopreventive activity in other cancer model systems is warranted.
Assuntos
Triterpenos Pentacíclicos/farmacologia , Perilla frutescens/química , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/prevenção & controle , Acetato de Tetradecanoilforbol/farmacologia , Animais , Interações Medicamentosas , Feminino , Camundongos , Camundongos Endogâmicos ICR , Triterpenos Pentacíclicos/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Transdução de Sinais , Neoplasias Cutâneas/patologiaRESUMO
The scalable syntheses of four oxygenated triterpenes have been implemented to access substantial quantities of maslinic acid, 3-epi-maslinic acid, corosolic acid, and 3-epi-corosolic acid. Semi-syntheses proceed starting from the natural products oleanolic acid and ursolic acid. Proceeding over five steps, each of the four compounds can be synthesized on the gram scale. Divergent diastereoselective reductions of α-hydroxy ketones provided access to the four targeted diol containing compounds from two precursors of the oleanane or ursane lineage. These compounds were subsequently evaluated for their ability to inhibit inflammatory gene expression in a mouse model of chemically induced skin inflammation. All compounds possessed the ability to inhibit the expression of one or more inflammatory genes induced by 12-O-tetradecanoylphorbol-13 acetate in mouse skin, however, three of the compounds, corosolic acid, 3-epi-corosolic acid and maslinic acid were more effective than the others. The availability of gram quantities will allow further testing of these compounds for potential anti-inflammatory activities as well as cancer chemopreventive activity.
Assuntos
Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/farmacologia , Citocinas/antagonistas & inibidores , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/farmacologia , Oxigênio/química , Pele/efeitos dos fármacos , Triterpenos/química , Triterpenos/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Citocinas/genética , Feminino , Camundongos , Camundongos Endogâmicos , Conformação Molecular , Ácido Oleanólico/síntese química , Ácido Oleanólico/química , Pele/metabolismo , Triterpenos/síntese química , Ácido UrsólicoRESUMO
In this study, the effects of combining ursolic acid + resveratrol, for possible combined inhibitory effects on skin tumor promotion, were evaluated. Ursolic acid, resveratrol, and the combination of ursolic acid + resveratrol were applied topically prior to 12-O-tetracanoylphorbol-13-acetate (TPA) treatment on mouse skin to examine their effect on TPA-induced signaling pathways, epidermal hyperproliferation, skin inflammation, inflammatory gene expression, and skin tumor promotion. The combination of ursolic acid + resveratrol produced a greater inhibition of TPA-induced epidermal hyperproliferation. The combination of ursolic acid + resveratrol inhibited TPA-induced signaling pathways, including EGFR, STAT3, Src, Akt, Cox-2, Fas, NF-κB, p38 MAPK, c-Jun, and JNK1/2 while increasing levels of tumor suppressors, such as p21 and PDCD4, to a greater extent compared with the groups treated with the individual compounds. Ursolic acid + resveratrol also induced a dramatic increase of p-AMPK-α(Thr172). Combined treatment with ursolic acid + resveratrol resulted in a greater inhibition of expression of proinflammatory cytokines, including Il1a, Il1b, and Il22. Furthermore, NF-κB, Egr-1, and AP-1 DNA binding activities after TPA treatment were dramatically decreased by the combination of ursolic acid + resveratrol. Treatment with ursolic acid + resveratrol during skin tumor promotion with TPA produced greater inhibition of tumor multiplicity and tumor size than with either agent alone. Collectively, the greater ability of the combination of ursolic acid + resveratrol to inhibit skin tumor promotion was due to the greater inhibitory effects on growth factor and inflammatory signaling, skin inflammation, and epidermal hyperproliferation induced by TPA treatment.
Assuntos
Anticarcinógenos/administração & dosagem , Neoplasias Cutâneas/induzido quimicamente , Neoplasias Cutâneas/tratamento farmacológico , Estilbenos/administração & dosagem , Acetato de Tetradecanoilforbol/química , Triterpenos/administração & dosagem , Animais , Carcinogênese , Núcleo Celular/metabolismo , Proliferação de Células , Citosol/metabolismo , Feminino , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ligação Proteica , Resveratrol , Transdução de Sinais/efeitos dos fármacos , Pele/patologia , Neoplasias Cutâneas/metabolismo , Ácido UrsólicoRESUMO
Chiral building blocks are valuable intermediates in the syntheses of natural products and pharmaceuticals. A scalable chemoenzymatic route to chiral diketides has been developed that includes the general synthesis of α-substituted, ß-ketoacyl N-acetylcysteamine thioesters followed by a biocatalytic cycle in which a glucose-fueled NADPH-regeneration system drives reductions catalyzed by isolated modular polyketide synthase (PKS) ketoreductases (KRs). To identify KRs that operate as active, stereospecific biocatalysts, 11 isolated KRs were incubated with 5 diketides and their products were analyzed by chiral chromatography. KRs that naturally reduce small polyketide intermediates were the most active and stereospecific toward the panel of diketides. Several biocatalytic reactions were scaled up to yield more than 100 mg of product. These syntheses demonstrate the ability of PKS enzymes to economically and greenly generate diverse chiral building blocks on a preparative scale.