RESUMO
A series of novel acylsulfonamide, acylsulfamide, and sulfonylurea bioisosteres of carboxylic acids were prepared as CXCR2 antagonists. Structure-activity relationships are reported for these series. One potent orally bioavailable inhibitor had excellent PK properties and was active in a lung injury model in hyperoxia-exposed newborn rats.
Assuntos
Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Pulmão/efeitos dos fármacos , Receptores de Interleucina-8B/antagonistas & inibidores , Sulfonamidas/química , Ácidos Sulfônicos/química , Compostos de Sulfonilureia/química , Administração Oral , Animais , Animais Recém-Nascidos , Disponibilidade Biológica , Lavagem Broncoalveolar , Ácidos Carboxílicos/farmacocinética , Quimiotaxia/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Hiperóxia , Pulmão/metabolismo , Lesão Pulmonar , Estrutura Molecular , Neutrófilos/metabolismo , Coelhos , Ratos , Relação Estrutura-AtividadeRESUMO
A series of 3,4,6-substituted 2-quinolones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). The fully optimized compound, 4-(4-ethyl-phenyl)-3-(2-methyl-3H-imidazol-4-yl)-2-quinolone-6-carbonitrile 21b, has an IC(50) of 2.5 nM in an in vitro assay and 5.0 nM in a bone marrow-derived macrophage cellular assay. Inhibition of FMS signaling in vivo was also demonstrated in a mouse pharmacodynamic model.
Assuntos
Macrófagos/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Quinolonas/síntese química , Quinolonas/farmacologia , Receptor de Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Proliferação de Células/efeitos dos fármacos , Polarização de Fluorescência , Genes fos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Quinolonas/farmacocinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/metabolismo , Relação Estrutura-AtividadeRESUMO
DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.
Assuntos
DNA Ligases/metabolismo , Medições Luminescentes/métodos , Hibridização de Ácido Nucleico/métodos , Staphylococcus aureus/enzimologia , Acridinas/química , Acridinas/metabolismo , Catálise , Clonagem Molecular , DNA Ligases/biossíntese , DNA Ligases/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Hidrólise , Cinética , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Projetos de Pesquisa/normas , Coloração e Rotulagem/métodos , Staphylococcus aureus/genética , Especificidade por SubstratoRESUMO
YihA has previously been characterized as an essential gene of unknown function in both Escherichia coli and Bacillus subtilis. It is conserved in bacteria and represents an attractive target for the discovery of new antibiotics. YihA encodes a putative GTP-binding protein. We have cloned and overexpressed the gene encoding E. coli YihA and initiated biochemical studies as a first step towards understanding its biological function. We showed by circular dichroism that the purified protein has a secondary structure typical of most GTP-binding proteins. It binds guanine nucleotides specifically, as demonstrated by fluorescence resonance energy transfer between 2'-(or-3')-O-(N-methylanthraniloyl) nucleotides (mant-nucleotides) and the tryptophans of YihA. The K(d) values for GDP and GTP were determined by competition with 2'-(or-3')-O-(N-methylanthraniloyl) GDP to be 3 and 27 microM, respectively. Using mutants of YihA we show that nucleotide binding occurs at the putative GTP-binding domain predicted from the primary sequence.
Assuntos
Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Proteínas de Ligação ao GTP/isolamento & purificação , Proteínas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Temperatura Alta , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Desnaturação Proteica , Alinhamento de SequênciaRESUMO
Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, DeltaH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.