RESUMO
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.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Astrocitoma/tratamento farmacológico , Benzimidazóis/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Isocitrato Desidrogenase/antagonistas & inibidores , Isocitrato Desidrogenase/genética , Compostos de Anilina/química , Compostos de Anilina/farmacocinética , Compostos de Anilina/toxicidade , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Astrocitoma/enzimologia , Astrocitoma/genética , Benzimidazóis/química , Benzimidazóis/farmacocinética , Benzimidazóis/toxicidade , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/toxicidade , Escherichia coli , Feminino , Glutaratos/metabolismo , Células HEK293 , Humanos , Isocitrato Desidrogenase/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sarcoma/tratamento farmacológico , Sarcoma/enzimologia , Sarcoma/genética , Células Sf9 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: IDH mutations frequently occur in diffuse gliomas and result in a neo-enzymatic activity that results in reduction of α-ketoglutarate to D-2-hydroxyglutarate. In gliomas, the frequency of IDH1 mutations in codon 132 increases in the order R132L-R132S-R132G-R132C-R132H with R132H constituting more than 90% of all IDH1 mutations. RESULTS: We determined the levels of D-2-hydroxyglutarate in glioma tissues with IDH1 mutations. D-2-hydroxyglutarate levels increased in the order of R132H-R132C-R132S/R132G/R132L. We expressed and purified IDH1 wild type and mutant protein for biochemical characterization. Enzyme kinetics of mutant IDH protein correlated well with D-2-hydroxyglutarate production in cells with R132H exhibiting the highest and R132L the lowest KM for α-ketoglutarate. Addition of D-2-hydroxyglutarate to the medium of cell lines revealed an inhibitory effect at higher concentrations. Migration of LN229 increased at lower D-2-hydroxyglutarate concentrations while higher concentrations showed no effect. CONCLUSION: These findings may suggest natural selection against the rare IDH1R132 mutations in human glioma due to toxicity caused by high levels of D-2-hydroxyglutarate.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Glutaratos/metabolismo , Isocitrato Desidrogenase/genética , Mutação/genética , Neoplasias Encefálicas/metabolismo , Movimento Celular/genética , Sobrevivência Celular/genética , Glioma/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Isocitrato Desidrogenase/metabolismo , Mutagênese/genética , Proteínas Mutantes , Transfecção , Ensaio Tumoral de Célula-TroncoRESUMO
Levels of (D)-2-hydroxyglutarate [D2HG, (R)-2-hydroxyglutarate] are increased in some metabolic diseases and in neoplasms with mutations in the isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) genes. Determination of D2HG is of relevance to diagnosis and monitoring of disease. Standard detection methods of D2HG levels are liquid-chromatography-mass spectrometry or gas-chromatography-mass spectrometry. Here we present a rapid, inexpensive and sensitive enzymatic assay for the detection of D2HG levels. The assay is based on the conversion of D2HG to α-ketoglutarate (αKG) in the presence of the enzyme (D)-2-hydroxyglutarate dehydrogenase (HGDH) and nicotinamide adenine dinucleotide (NAD(+)). Determination of D2HG concentration is based on the detection of stoichiometrically generated NADH. The quantification limit of the enzymatic assay for D2HG in tumor tissue is 0.44 µM and in serum 2.77 µM. These limits enable detection of basal D2HG levels in human tumor tissues and serum without IDH mutations. Levels of D2HG in frozen and paraffin-embedded tumor tissues containing IDH mutations or in serum from acute myeloid leukemia patients with IDH mutations are significantly higher and can be easily identified with this assay. In conclusion, the assay presented is useful for differentiating basal from elevated D2HG levels in tumor tissue, serum, urine, cultured cells and culture supernatants.