RESUMO
Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies1. Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies2-4. Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2+ breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Feminino , Humanos , Camundongos , Receptor ErbB-2/genéticaRESUMO
BACKGROUND: There are two FDA-approved anti-CD38 monoclonal antibodies for treatment of multiple myeloma: isatuximab and daratumumab. Owing to expression of CD38 on reagent red blood cells (RBCs), these antibodies interfere with indirect antiglobulin tests (IATs). We sought to understand differences in such interference by performing binding experiments. STUDY DESIGN AND METHODS: In vitro experiments to compare the binding to RBCs of isatuximab and daratumumab alone or in the presence of a mouse anti-human CD38 antibody (HB-7 or AT13/5) or a nicotinamide adenine dinucleotide-analog CD38 inhibitor were performed and quantified by flow cytometry, imaging, mass spectrometry, surface plasmon resonance, and LigandTracer technologies. Serologic testing was performed on plasma samples spiked with isatuximab or daratumumab. RESULTS: CD38 expressed on RBCs can be directly bound by daratumumab, whereas isatuximab requires a co-factor, such as HB-7, AT13/5, or a CD38 inhibitor, suggesting that the isatuximab epitope on RBCs is masked in vitro. Daratumumab samples more frequently showed interference and had stronger reactions than isatuximab samples. Dithiothreitol treatment was equally effective in mitigating the interference caused by either drug. DISCUSSION: Both isatuximab and daratumumab interfere with IATs but at different magnitudes, reflecting distinct binding to CD38 on RBCs. From the binding studies, we conclude that the isatuximab epitope on RBCs is masked in vitro and binding requires a certain CD38 conformation or co-factor. This circumstance may explain why interference is seen only in a subset of patients receiving isatuximab when compared with interference seen in most patients on daratumumab therapy.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Neuroblastoma , Camundongos , Animais , ADP-Ribosil Ciclase 1 , Mapeamento de Epitopos , Anticorpos Monoclonais , Mieloma Múltiplo/terapia , Antineoplásicos/uso terapêutico , Neuroblastoma/tratamento farmacológico , EpitoposRESUMO
Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg(2+). A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Isocitrato Desidrogenase/química , Neoplasias/tratamento farmacológico , Sítio Alostérico , Cristalografia por Raios X , Metilação de DNA/genética , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Escherichia coli , Regulação Neoplásica da Expressão Gênica , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/biossíntese , Isocitrato Desidrogenase/genética , Magnésio/química , Proteínas Mutantes/química , Proteínas Mutantes/genética , Neoplasias/genética , Neoplasias/patologia , Conformação ProteicaRESUMO
PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.
Assuntos
Proteínas de Neoplasias , Neoplasias , Inibidores de Proteínas Quinases , Ribonucleoproteína Nuclear Pequena U4-U6 , Linhagem Celular Tumoral , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Ribonucleoproteína Nuclear Pequena U4-U6/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismoRESUMO
Human cytomegalovirus (CMV) is a large enveloped virus that encodes multiple glycoproteins required for virus-cell binding and fusion. To assess the binding properties of antibodies with target glycoprotein in a natural context of infection, we investigated the feasibility of using the surface plasmon resonance (SPR) technique for studying the direct binding of antibodies with CMV virions. Direct immobilization of whole virions to sensor surface and a surface regeneration procedure allowed for quantitative and reproducible measurements of binding affinity and binding kinetics of antibody-whole virion interactions. The conformational and functional integrity of viral particles was not compromised by the regeneration condition as evaluated with antibodies recognizing conformational epitopes and by electron microscopy. Binding of an irrelevant antibody was not observed, indicating the high specificity of the method. A panel of anti-gB antibodies was measured and the binding affinities correlated fairly well with those determined by ELISA. These data demonstrated that the interaction of anti-gB antibody with whole virion of large enveloped CMV can be quantitatively studied using SPR. This method has been successfully applied for screening and selection of anti-CMV antibodies and can be potentially extended to study antibody-glycoprotein interactions of other related herpesviruses.
Assuntos
Anticorpos Antivirais/imunologia , Citomegalovirus/imunologia , Ressonância de Plasmônio de Superfície/métodos , Proteínas do Envelope Viral/imunologia , Especificidade de Anticorpos/imunologia , Citomegalovirus/ultraestrutura , Ensaio de Imunoadsorção Enzimática , Humanos , Cinética , Reprodutibilidade dos Testes , Propriedades de Superfície , Vírion/ultraestruturaRESUMO
Undecaprenyl pyrophosphate synthase (UPPS) catalyzes the consecutive condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate the C(55) undecaprenyl pyrophosphate (UPP). It has been demonstrated that tetramic acids (TAs) are selective and potent inhibitors of UPPS, but the mode of inhibition was unclear. In this work, we used a fluorescent FPP probe to study possible TA binding at the FPP binding site. A photosensitive TA analogue was designed and synthesized for the study of the site of interaction of TA with UPPS using photo-cross-linking and mass spectrometry. The interaction of substrates with UPPS and with the UPPS.TA complex was investigated by protein fluorescence spectroscopy. Our results suggested that tetramic acid binds to UPPS at an allosteric site adjacent to the FPP binding site. TA binds to free UPPS enzyme but not to substrate-bound UPPS. Unlike Escherichia coli UPPS which follows an ordered substrate binding mechanism, Streptococcus pneumoniae UPPS appears to follow a random-sequential substrate binding mechanism. Only one substrate, FPP or IPP, is able to bind to the UPPS.TA complex, but the quaternary complex, UPPS.TA.FPP.IPP, cannot be formed. We propose that binding of TA to UPPS significantly alters the conformation of UPPS needed for proper substrate binding. As the result, substrate turnover is prevented, leading to the inhibition of UPPS catalytic activity. These probe compounds and biophysical assays also allowed us to quickly study the mode of inhibition of other UPPS inhibitors identified from a high-throughput screening and inhibitors produced from a medicinal chemistry program.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirrolidinonas/farmacologia , Alquil e Aril Transferases/química , Alquil e Aril Transferases/isolamento & purificação , Alquil e Aril Transferases/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biofísica , Escherichia coli/enzimologia , Corantes Fluorescentes , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Pirrolidinonas/antagonistas & inibidores , Espectrometria de Massas por Ionização por Electrospray , Streptococcus pneumoniae/enzimologiaRESUMO
Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy.
Assuntos
Anticorpos Biespecíficos , Mieloma Múltiplo , Animais , Anticorpos Biespecíficos/uso terapêutico , Antígenos CD28 , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Receptores de Antígenos de Linfócitos T , Linfócitos TRESUMO
CD73/Ecto-5'-nucleotidase is a membrane-tethered ecto-enzyme that works in tandem with CD39 to convert extracellular adenosine triphosphate (ATP) into adenosine. CD73 is highly expressed on various types of cancer cells and on infiltrating suppressive immune cells, leading to an elevated concentration of adenosine in the tumor microenvironment, which elicits a strong immunosuppressive effect. In preclinical studies, targeting CD73 with anti-CD73 antibody results in favorable antitumor effects. Despite initial studies using antibodies, inhibition of CD73 catalytic activity using small-molecule inhibitors may be more effective in lowering extracellular adenosine due to better tumor penetration and distribution. To screen small-molecule libraries, we explored multiple approaches, including colorimetric and fluorescent biochemical assays, and due to some limitations with these assays, we developed a mass spectrometry (MS)-based assay. Only the MS-based assay offers the sensitivity and dynamic range required for screening small-molecule libraries at a substrate concentration close to the Km value of substrate and for evaluating the mode of binding of screening hits. To achieve a throughput suitable for high-throughput screening (HTS), we developed a RapidFire-tandem mass spectrometry (RF-MS/MS)-based multiplex assay. This assay allowed a large diverse compound library to be screened at a speed of 1536 reactions per 40-50 min.
Assuntos
5'-Nucleotidase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Bioensaio/métodos , Linhagem Celular , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Células HEK293 , Humanos , Camundongos , Espectrometria de Massas em Tandem/métodosRESUMO
Tumor cells display fundamental changes in metabolism and nutrient uptake in order to utilize additional nutrient sources to meet their enhanced bioenergetic requirements. Glutamine (Gln) is one such nutrient that is rapidly taken up by tumor cells to fulfill this increased metabolic demand. A vital step in the catabolism of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase (GLS). This study has identified GLS a potential therapeutic target in breast cancer, specifically in the basal subtype that exhibits a deregulated glutaminolysis pathway. Using inducible shRNA mediated gene knockdown, we discovered that loss of GLS function in triple-negative breast cancer (TNBC) cell lines with a deregulated glutaminolysis pathway led to profound tumor growth inhibition in vitro and in vivo. GLS knockdown had no effect on growth and metabolite levels in non-TNBC cell lines. We rescued the anti-tumor effect of GLS knockdown using shRNA resistant cDNAs encoding both GLS isoforms and by addition of an α-ketoglutarate (αKG) analog thus confirming the critical role of GLS in TNBC. Pharmacological inhibition of GLS with the small molecule inhibitor CB-839 reduced cell growth and led to a decrease in mammalian target of rapamycin (mTOR) activity and an increase in the stress response pathway driven by activating transcription factor 4 (ATF4). Finally, we found that GLS inhibition synergizes with mTOR inhibition, which introduces the possibility of a novel therapeutic strategy for TNBC. Our study revealed that GLS is essential for the survival of TNBC with a deregulated glutaminolysis pathway. The synergistic activity of GLS and mTOR inhibitors in TNBC cell lines suggests therapeutic potential of this combination for the treatment of vulnerable subpopulations of TNBC.
Assuntos
Glutaminase/metabolismo , Glutamina/metabolismo , Serina-Treonina Quinases TOR/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/enzimologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The development of an effective AIDS vaccine has been challenging because of viral genetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs). We engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to those of human bnAbs, and conferred complete immunity against a mixture of simian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and they may be applicable for treatment of diverse diseases, including infections, cancer, and autoimmunity.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/farmacocinética , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Antígenos CD4/imunologia , Cristalografia por Raios X , Anticorpos Anti-HIV/administração & dosagem , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Humanos , Macaca mulatta , Engenharia de Proteínas , Síndrome de Imunodeficiência Adquirida dos Símios/sangueRESUMO
Mass spectrometry (MS) has been applied to drug discovery for many years. With the advent of new ionization techniques, MS has emerged as an important analytical tool in identification and characterization of protein targets, structure elucidation of synthetic compounds, and early drug metabolism and pharmacokinetics studies. Two MS-based strategies, function-based and affinity-based, have been employed in recent years for screening and evaluation of compounds. In the function-based approach, the effects of compounds on the biological activity of a target molecule are measured. In the affinity-based approach, compounds are screened based on their binding affinities to target molecules. The interaction between targets and compounds can be directly evaluated by monitoring the formation of non-covalent target-ligand complexes (direct detection) or indirectly evaluated by detecting the compounds after separating bound compounds from unbound (indirect detection). Various techniques including high performance liquid chromatography (HPLC)-MS, size exclusion chromatography (SEC)-MS, frontal affinity chromatography (FAC)-MS and desorption/ionization on silicon (DIOS)-MS can be applied. The recent advances, relative advantages, and limitations of each MS-based method as a tool in compound screening and compound evaluation in the early stages of drug discovery are discussed in this review.
Assuntos
Desenho de Fármacos , Espectrometria de Massas , Farmacologia/instrumentação , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Relação Estrutura-AtividadeRESUMO
Heterozygous mutation of IDH1 in cancers modifies IDH1 enzymatic activity, reprogramming metabolite flux and markedly elevating 2-hydroxyglutarate (2-HG). Here, we found that 2-HG depletion did not inhibit growth of several IDH1 mutant solid cancer types. To identify other metabolic therapeutic targets, we systematically profiled metabolites in endogenous IDH1 mutant cancer cells after mutant IDH1 inhibition and discovered a profound vulnerability to depletion of the coenzyme NAD+. Mutant IDH1 lowered NAD+ levels by downregulating the NAD+ salvage pathway enzyme nicotinate phosphoribosyltransferase (Naprt1), sensitizing to NAD+ depletion via concomitant nicotinamide phosphoribosyltransferase (NAMPT) inhibition. NAD+ depletion activated the intracellular energy sensor AMPK, triggered autophagy, and resulted in cytotoxicity. Thus, we identify NAD+ depletion as a metabolic susceptibility of IDH1 mutant cancers.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glioblastoma/tratamento farmacológico , Isocitrato Desidrogenase/genética , Mutação , NAD/deficiência , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Metabolismo Energético/efeitos dos fármacos , Ativação Enzimática , Feminino , Glioblastoma/enzimologia , Glioblastoma/genética , Glioblastoma/patologia , Glutaratos/metabolismo , Células HEK293 , Humanos , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/metabolismo , Metabolômica/métodos , Camundongos SCID , Terapia de Alvo Molecular , Nicotinamida Fosforribosiltransferase/metabolismo , Pentosiltransferases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Peptídeo Sintases/antagonistas & inibidores , Peptídeo Sintases/metabolismo , Cromatografia Líquida/métodos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , Cinética , Espectrometria de Massas/métodos , Sensibilidade e Especificidade , Especificidade por SubstratoRESUMO
Clostridium difficile (C. difficile) is a Gram positive, anaerobic bacterium that infects the lumen of the large intestine and produces toxins. This results in a range of syndromes from mild diarrhea to severe toxic megacolon and death. Alarmingly, the prevalence and severity of C. difficile infection are increasing; thus, associated morbidity and mortality rates are rising. 4-Aminothiazolyl analogues of the antibiotic natural product GE2270 A (1) were designed, synthesized, and optimized for the treatment of C. difficile infection. The medicinal chemistry effort focused on enhancing aqueous solubility relative to that of the natural product and previous development candidates (2, 3) and improving antibacterial activity. Structure-activity relationships, cocrystallographic interactions, pharmacokinetics, and efficacy in animal models of infection were characterized. These studies identified a series of dicarboxylic acid derivatives, which enhanced solubility/efficacy profile by several orders of magnitude compared to previously studied compounds and led to the selection of LFF571 (4) as an investigational new drug for treating C. difficile infection.
Assuntos
Antibacterianos/síntese química , Clostridioides difficile/efeitos dos fármacos , Enterocolite Pseudomembranosa/tratamento farmacológico , Tiazóis/síntese química , Animais , Antibacterianos/farmacocinética , Antibacterianos/farmacologia , Cricetinae , Cristalografia por Raios X , Enterococcus/efeitos dos fármacos , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Feminino , Masculino , Mesocricetus , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Fator Tu de Elongação de Peptídeos/antagonistas & inibidores , Fator Tu de Elongação de Peptídeos/química , Ratos , Ratos Sprague-Dawley , Solubilidade , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pyogenes/efeitos dos fármacos , Relação Estrutura-Atividade , Tiazóis/farmacocinética , ÁguaRESUMO
Argyrins, produced by myxobacteria and actinomycetes, are cyclic octapeptides with antibacterial and antitumor activity. Here, we identify elongation factor G (EF-G) as the cellular target of argyrin B in bacteria, via resistant mutant selection and whole genome sequencing, biophysical binding studies and crystallography. Argyrin B binds a novel allosteric pocket in EF-G, distinct from the known EF-G inhibitor antibiotic fusidic acid, revealing a new mode of protein synthesis inhibition. In eukaryotic cells, argyrin B was found to target mitochondrial elongation factor G1 (EF-G1), the closest homologue of bacterial EF-G. By blocking mitochondrial translation, argyrin B depletes electron transport components and inhibits the growth of yeast and tumor cells. Further supporting direct inhibition of EF-G1, expression of an argyrin B-binding deficient EF-G1 L693Q variant partially rescued argyrin B-sensitivity in tumor cells. In summary, we show that argyrin B is an antibacterial and cytotoxic agent that inhibits the evolutionarily conserved target EF-G, blocking protein synthesis in bacteria and mitochondrial translation in yeast and mammalian cells.
Assuntos
Oligopeptídeos/metabolismo , Fator G para Elongação de Peptídeos/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Animais , Burkholderia/efeitos dos fármacos , Linhagem Celular Tumoral , Sequência Conservada , Cristalografia por Raios X , Humanos , Mamíferos , Testes de Sensibilidade Microbiana , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Fator G para Elongação de Peptídeos/antagonistas & inibidores , Fator G para Elongação de Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Three cyclophilin inhibitors (DEBIO-025, SCY635, and NIM811) are currently in clinical trials for hepatitis C therapy. The mechanism of action of these, however, is not completely understood. There are at least 16 cyclophilins expressed in human cells which are involved in a diverse set of cellular processes. Large-scale siRNA experiments, chemoproteomic assays with cyclophilin binding compounds, and mRNA profiling of HCV replicon containing cells were used to identify the cyclophilins that are instrumental to HCV replication. The previously reported cyclophilin A was confirmed and additional cyclophilin containing pathways were identified. Together, the experiments provide strong evidence that NIM811 reduces viral replication by inhibition of multiple cyclophilins and pathways with protein trafficking as the most strongly and persistently affected pathway.
Assuntos
Ciclofilinas/metabolismo , Hepacivirus/fisiologia , Interações Hospedeiro-Patógeno , Replicação Viral , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Ciclosporina/química , Ciclosporina/farmacologia , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Modelos Biológicos , Estrutura Molecular , Proteoma/análise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Ubiquitin plays essential roles in various cellular processes; therefore, it is of keen interest to study the structure-function relationship of ubiquitin itself. We investigated the modification of Lys(6) of ubiquitin and its physiological consequences. Mass spectrometry-based peptide mapping and N-terminal sequencing demonstrated that, of the 7 Lys residues in ubiquitin, Lys(6) was the most readily labeled with sulfosuccinimidobiotin. Lys(6)-biotinylated ubiquitin was incorporated into high molecular mass ubiquitin conjugates as efficiently as unmodified ubiquitin. However, Lys(6)-biotinylated ubiquitin inhibited ubiquitin-dependent proteolysis, as conjugates formed with Lys(6)-biotinylated ubiquitin were resistant to proteasomal degradation. Ubiquitins with a mutation of Lys(6) had similar phenotypes as Lys(6)-biotinylated ubiquitin. Lys(6) mutant ubiquitins (K6A, K6R, and K6W) also inhibited ATP-dependent proteolysis and caused accumulation of ubiquitin conjugates. Conjugates formed with K6W mutant ubiquitin were also resistant to proteasomal degradation. The dominant-negative effect of Lys(6)-modified ubiquitin was further demonstrated in intact cells. Overexpression of K6W mutant ubiquitin resulted in accumulation of intracellular ubiquitin conjugates, stabilization of typical substrates for ubiquitin-dependent proteolysis, and enhanced susceptibility to oxidative stress. Taken together, these results show that Lys(6)-modified ubiquitin is a potent and specific inhibitor of ubiquitin-mediated protein degradation.