RESUMO
Libraries of nonpurified resorcinol amide derivatives were screened by surface plasmon resonance (SPR) to determine the binding dissociation constant (off-rate, kd) for compounds binding to the pyruvate dehydrogenase kinase (PDHK) enzyme. Parallel off-rate measurements against HSP90 and application of structure-based drug design enabled rapid hit to lead progression in a program to identify pan-isoform ATP-competitive inhibitors of PDHK. Lead optimization identified selective sub-100-nM inhibitors of the enzyme which significantly reduced phosphorylation of the E1α subunit in the PC3 cancer cell line in vitro.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Linhagem Celular Tumoral , Desenho de Fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Masculino , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de AcetilRESUMO
Pyruvate dehydrogenase kinase (PDK) is a pivotal enzyme in cellular energy metabolism that has previously been implicated in cancer through both RNAi based studies and clinical correlations with poor prognosis in several cancer types. Here, we report the discovery of a novel and selective ATP competitive pan-isoform inhibitor of PDK, VER-246608. Consistent with a PDK mediated MOA, VER-246608 increased pyruvate dehydrogenase complex (PDC) activity, oxygen consumption and attenuated glycolytic activity. However, these effects were only observed under D-glucose-depleted conditions and required almost complete ablation of PDC E1α subunit phosphorylation. VER-246608 was weakly anti-proliferative to cancer cells in standard culture media; however, depletion of either serum or combined D-glucose/L-glutamine resulted in enhanced cellular potency. Furthermore, this condition-selective cytostatic effect correlated with reduced intracellular pyruvate levels and an attenuated compensatory response involving deamination of L-alanine. In addition, VER-246608 was found to potentiate the activity of doxorubicin. In contrast, the lipoamide site inhibitor, Nov3r, demonstrated sub-maximal inhibition of PDK activity and no evidence of cellular activity. These studies suggest that PDK inhibition may be effective under the nutrient-depleted conditions found in the tumour microenvironment and that combination treatments should be explored to reveal the full potential of this therapeutic strategy.
Assuntos
Trifosfato de Adenosina/metabolismo , Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinas/farmacologia , Ligação Competitiva , Inibidores Enzimáticos/química , Glicólise/efeitos dos fármacos , Humanos , Isoenzimas , Células K562 , Modelos Moleculares , Estrutura Molecular , Invasividade Neoplásica , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Células Tumorais CultivadasRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease that is characterized by a particularly marked resistance to chemotherapy. We previously showed an association between decreased expression of BNIP3 and chemoresistance in PDAC cell lines. To further explore the molecular basis of chemoresistance in PDAC, we analyzed microarray data obtained from normal pancreas and PDAC tumor samples to identify genes exhibiting a negative correlation with the expression profile of BNIP3. This analysis identified several S100 family proteins, of which two, S100A2 and S100A4, showed in vitro the ability to repress exogenous BNIP3 promoter activity. We subsequently showed that RNA interference-mediated S100A4 knockdown resulted in an elevated expression of BNIP3 in PDAC cell lines that possess an unmethylated BNIP3 promoter, suggesting that, in addition to hypermethylation, S100A4 overexpression may represent an alternative mechanism for inhibiting BNIP3 function in PDAC. S100A4 knockdown also resulted in an increased sensitivity of PDAC cell lines to gemcitabine treatment, which was coupled with an increase in apoptosis and cell cycle arrest. To investigate the underlying mechanisms mediating these effects, we studied the effect of silencing the expression of S100A4 on the induction of apoptosis, cell cycle arrest, and the activation of apoptotic mediators. Knockdown of S100A4 clearly induced apoptosis with increased fragmentation of DNA and phosphatidyl serine externalization; activation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase; and release of cytochrome c into the cytosol. These findings provide evidence that supports a novel role for S100A4 as a prosurvival factor in pancreatic cancer.
Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/fisiologia , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas S100/fisiologia , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Ativação Enzimática , Humanos , Proteínas de Membrana/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/metabolismo , Fatores de Tempo , Transfecção , GencitabinaRESUMO
Hypoxia-inducible factor 1 (HIF-1) functions as a master regulator of oxygen homeostasis in metazoan species. HIF-1 mediates changes in gene transcription in response to changes in cellular oxygenation. The half-life of the HIF-1alpha subunit is determined by oxygen-dependent prolyl hydroxylation, which is required for binding of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase that targets HIF-1alpha for ubiquitination and degradation. Here, we demonstrate that OS-9, the protein product of a widely expressed gene, interacts with both HIF-1alpha and HIF-1alpha prolyl hydroxylases. OS-9 gain-of-function promotes HIF-1alpha hydroxylation, VHL binding, proteasomal degradation of HIF-1alpha, and inhibition of HIF-1-mediated transcription. OS-9 loss-of-function caused by RNA interference increases HIF-1alpha protein levels, HIF-1-mediated transcription, and VEGF mRNA expression under nonhypoxic conditions. These data indicate that OS-9 is an essential component of a multiprotein complex that regulates HIF-1alpha levels in an O2-dependent manner.