RESUMEN
The oncometabolite (R)-2-hydroxyglutarate (R-2-HG) produced by isocitrate dehydrogenase (IDH) mutations promotes gliomagenesis via DNA and histone methylation. Here, we identify an additional activity of R-2-HG: tumor cell-derived R-2-HG is taken up by T cells where it induces a perturbation of nuclear factor of activated T cells transcriptional activity and polyamine biosynthesis, resulting in suppression of T cell activity. IDH1-mutant gliomas display reduced T cell abundance and altered calcium signaling. Antitumor immunity to experimental syngeneic IDH1-mutant tumors induced by IDH1-specific vaccine or checkpoint inhibition is improved by inhibition of the neomorphic enzymatic function of mutant IDH1. These data attribute a novel, non-tumor cell-autonomous role to an oncometabolite in shaping the tumor immune microenvironment.
Asunto(s)
Glutaratos/metabolismo , Inmunidad , Linfocitos T/inmunología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/inmunología , Calcio/metabolismo , Línea Celular Tumoral , Proliferación Celular , Glioma/genética , Glioma/inmunología , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Mutación/genética , Factores de Transcripción NFATC/metabolismo , Comunicación Paracrina , Poliaminas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
Mutations in codon 132 of isocitrate dehydrogenase (IDH) 1 are frequent in diffuse glioma, acute myeloid leukemia, chondrosarcoma and intrahepatic cholangiocarcinoma. These mutations result in a neomorphic enzyme specificity which leads to a dramatic increase of intracellular D-2-hydroxyglutarate (2-HG) in tumor cells. Therefore, mutant IDH1 protein is a highly attractive target for inhibitory drugs. Here, we describe the development and properties of BAY 1436032, a pan-inhibitor of IDH1 protein with different codon 132 mutations. BAY 1436032 strongly reduces 2-HG levels in cells carrying IDH1-R132H, -R132C, -R132G, -R132S and -R132L mutations. Cells not carrying IDH mutations were unaffected. BAY 1436032 did not exhibit toxicity in vitro or in vivo. The pharmacokinetic properties of BAY 1436032 allow for oral administration. In two independent experiments, BAY 1436032 has been shown to significantly prolong survival of mice intracerebrally transplanted with human astrocytoma carrying the IDH1R132H mutation. In conclusion, we developed a pan-inhibitor targeting tumors with different IDH1R132 mutations.
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Astrocitoma/tratamiento farmacológico , Bencimidazoles/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/genética , Compuestos de Anilina/química , Compuestos de Anilina/farmacocinética , Compuestos de Anilina/toxicidad , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Astrocitoma/enzimología , Astrocitoma/genética , Bencimidazoles/química , Bencimidazoles/farmacocinética , Bencimidazoles/toxicidad , Neoplasias Encefálicas/enzimología , Neoplasias Encefálicas/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Escherichia coli , Femenino , Glutaratos/metabolismo , Células HEK293 , Humanos , Isocitrato Deshidrogenasa/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcoma/tratamiento farmacológico , Sarcoma/enzimología , Sarcoma/genética , Células Sf9 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Penicillin-resistance in Streptococcus pneumoniae is mainly due to alterations in genes encoding the target enzymes for beta-lactams, the penicillin-binding proteins (PBPs). However, non-PBP genes are altered in beta-lactam-resistant laboratory mutants and confer decreased susceptibility to beta-lactam antibiotics. Two piperacillin resistant laboratory mutants of Streptococcus pneumoniae R6 contain mutations in the putative glycosyltransferase gene cpoA. The CpoA gene is part of an operon including another putative glycosyltransferase gene spr0982, both of which being homologous to glycolipid synthases present in other Gram-positive bacteria. RESULTS: We now show that the cpoA mutants as well as a cpoA deletion mutant are defective in the synthesis of galactosyl-glucosyl-diacylglycerol (GalGlcDAG) in vivo consistent with the in vitro function of CpoA as α-GalGlcDAG synthase as shown previously. In addition, the proportion of phosphatidylglycerol increased relative to cardiolipin in cpoA mutants. Moreover, cpoA mutants are more susceptible to acidic stress, have an increased requirement for Mg(2+) at low pH, reveal a higher resistance to lysis inducing conditions and are hypersensitive to bacitracin. CONCLUSIONS: The data show that deficiency of the major glycolipid GalGlcDAG causes a pleitotropic phenotype of cpoA mutant cells consistent with severe membrane alterations. We suggest that the cpoA mutations selected with piperacillin are directed against the lytic response induced by the beta-lactam antibiotic.
Asunto(s)
Técnicas de Inactivación de Genes , Glicosiltransferasas/metabolismo , Metabolismo de los Lípidos , Lípidos/análisis , Mutación , Streptococcus pneumoniae/metabolismo , Antibacterianos/farmacología , Membrana Celular/metabolismo , Glicosiltransferasas/genética , Piperacilina/farmacología , Streptococcus pneumoniae/química , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Resistencia betalactámicaRESUMEN
The influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses.
Asunto(s)
Virus de la Influenza A/enzimología , Péptidos/química , Péptidos/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Línea Celular , Humanos , Virus de la Influenza A/metabolismo , Datos de Secuencia Molecular , Péptidos/síntesis química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Proteínas Virales/genéticaRESUMEN
To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFNα/ß receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses.