RESUMO
Intracellular levels of the hypoxia-inducible transcription factor (HIF) are regulated under normoxic conditions by prolyl hydroxylases (PHD1, 2, and 3). Treatment of cells with PHD inhibitors stabilizes HIF-1α, eliciting an artificial hypoxic response that includes the transcription of genes involved in erythropoiesis, angiogenesis, and glycolysis. The different in vivo roles of the three PHD isoforms are not yet known, making a PHD-selective inhibitor useful as a biological tool. Although several chemical series of PHD inhibitors have been described, significant isoform selectivity has not been reported. Here we report the synthesis and activity of dipeptidyl analogues derived from a potent but non-selective quinolone scaffold. The compounds were prepared by Pd-catalyzed reductive carbonylation of the 6-iodoquinolone derivative to form the aldehyde directly, which was then attached to a solid support via reductive amination. Amino acids were coupled, and the resulting dipeptidyl-quinolone derivatives were screened, revealing retention of PHD inhibitory activity but an altered PHD1, 2, and 3 selectivity profile. The compounds were found to be â¼10-fold more potent against PHD1 and PHD3 than against PHD2, whereas the specific parent compound had shown no appreciable selectivity among the different PHD isoforms.
Assuntos
Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Isoenzimas/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Quinolonas/farmacologia , Técnicas de Química Combinatória , Dipeptídeos/síntese química , Dipeptídeos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Estrutura Molecular , Pró-Colágeno-Prolina Dioxigenase/química , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Quinolonas/síntese química , Quinolonas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Fusobacterium nucleatum is a Gram-negative anaerobic rod found in dental plaque biofilms, and is an opportunistic pathogen implicated in periodontitis as well as a wide range of systemic abscesses and infections. Genomic analyses of F. nucleatum indicate considerable genetic diversity and a prominent role for horizontal gene transfer in the evolution of the species. Several plasmids isolated from F. nucleatum, including pFN1, harbor relaxase gene homologs that may function in plasmid mobilization. In this investigation we examined the RP4-mediated mobilization properties of pFN1 and the prevalence of pFN1-related sequences in a panel of F. nucleatum clinical isolates. The fusobacterial plasmid pFN1 was mobilized by RP4 at a high frequency. Deletion analyses were used to delineate the core mobilon of pFN1, which consisted of the relaxase gene (rlx), an upstream open reading frame ORF4 and a region of DNA upstream of ORF4 with potential nic sites. To examine the prevalence of pFN1 in a panel of clinical isolates, total DNA isolated from the strains was hybridized with pFN1 replication (repA) and rlx gene probes. DNA from strains harboring plasmids known to be homologous to pFN1 hybridized with both the repA and rlx probes. Five additional strains were rlx-positive but repA-negative, indicating a greater prevalence of rlx-related genes in comparison with repA-related genes. Plasmid or plasmid-related sequences were identified in 11.5% of the strains examined. These findings demonstrate mobilization properties of a fusobacterial plasmid that may be important in horizontal gene transfer.
Assuntos
Fusobacterium nucleatum/genética , Transferência Genética Horizontal , Plasmídeos/genética , Plasmídeos/metabolismo , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Análise de Sequência de DNARESUMO
We report the structure-based design and synthesis of a novel series of aza-benzimidazoles as PHD2 inhibitors. These efforts resulted in compound 22, which displayed highly potent inhibition of PHD2 function in vitro.
Assuntos
Compostos Aza/síntese química , Compostos Aza/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Compostos Aza/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Benzimidazóis/química , Técnicas de Química Combinatória , Cristalografia por Raios X , Humanos , Prolina Dioxigenases do Fator Induzível por Hipóxia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Inhibition of the PHD2 enzyme has been associated with increased red blood cell levels. From a screening hit, a series of novel hydroxyl-thiazoles were developed as potent PHD2 inhibitors.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/síntese química , Tiazóis/química , Química Farmacêutica/métodos , Diabetes Mellitus/tratamento farmacológico , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Eritrócitos/enzimologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Concentração Inibidora 50 , Modelos Químicos , Modelos Moleculares , Infarto do Miocárdio/tratamento farmacológico , Doenças Vasculares Periféricas/terapia , Acidente Vascular Cerebral/tratamento farmacológico , Relação Estrutura-Atividade , Tiazóis/farmacologiaRESUMO
Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.
Assuntos
Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Alemtuzumab , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Antineoplásicos/metabolismo , Linfócitos B/imunologia , Proteínas do Sistema Complemento/metabolismo , Citotoxicidade Imunológica , Variação Genética , Humanos , Técnicas In Vitro , Depleção Linfocítica , Macaca fascicularis , Engenharia de Proteínas , Receptores de IgG/metabolismo , TrastuzumabRESUMO
Tumor necrosis factor (TNF) is a key regulator of inflammatory responses and has been implicated in many pathological conditions. We used structure-based design to engineer variant TNF proteins that rapidly form heterotrimers with native TNF to give complexes that neither bind to nor stimulate signaling through TNF receptors. Thus, TNF is inactivated by sequestration. Dominant-negative TNFs represent a possible approach to anti-inflammatory biotherapeutics, and experiments in animal models show that the strategy can attenuate TNF-mediated pathology. Similar rational design could be used to engineer inhibitors of additional TNF superfamily cytokines as well as other multimeric ligands.