RESUMO
Two novel compounds, pyridopyrimidines (1) and naphthyridines (2) were identified as potent inhibitors of bacterial NAD(+)-dependent DNA ligase (Lig) A in a fragment screening. SAR was guided by molecular modeling and X-ray crystallography. It was observed that the diaminonitrile pharmacophore made a key interaction with the ligase enzyme, specifically residues Glu114, Lys291, and Leu117. Synthetic challenges limited opportunities for diversification of the naphthyridine core, therefore most of the SAR was focused on a pyridopyrimidine scaffold. The initial diversification at R(1) improved both enzyme and cell potency. Further SAR developed at the R(2) position using the Negishi cross-coupling reaction provided several compounds, among these compounds 22g showed good enzyme potency and cellular potency.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , DNA Ligases/antagonistas & inibidores , NAD/metabolismo , Naftiridinas/farmacologia , Pirimidinas/farmacologia , Antibacterianos/síntese química , Proteínas de Bactérias/química , DNA Ligases/química , Haemophilus influenzae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Naftiridinas/síntese química , Pirimidinas/síntese química , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Respiratory Syncytial Virus (RSV) is a major cause of lower respiratory tract infections with no effective treatment available. Finding novel inhibitors of RSV is an important first step towards developing an efficacious RSV therapy. Here we report the characterization of three novel classes of RSV replication inhibitors identified through a high throughput RSV replicon screen of â¼1million compounds in the AstraZeneca compound collection. These inhibitors, cpd 1, 2, and 3, specifically targeted RSV and were not active against other viruses tested. Resistance selection in RSV A2 with cpd 1 identified escape viruses with mutations mapped to the RSV L protein, an RNA-dependent RNA polymerase (Y1631C and I1413T). Recombinant RSV containing the L Y1631C substitution conferred resistance towards cpd 1, suggesting that the RSV polymerase is the target of this inhibitor. Interestingly, cpd 3, a nucleoside analog, induced a single resistant mutation in the P protein (D231V), indicating a novel mode of action not previously reported. cpd 2 affected host cell cycle and no frequent mutation was isolated following resistance selection, suggesting its possible involvement of a host-targeted mechanism. Taken together, we have identified three novel RSV inhibitors with different modes of action, providing new chemistry starting points for the discovery and development of future RSV therapeutic treatment.
Assuntos
Antivirais/química , Antivirais/farmacologia , Ensaios de Triagem em Larga Escala , Replicon/efeitos dos fármacos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Benzotiazóis/química , Benzotiazóis/farmacologia , Farmacorresistência Viral/genética , Indóis/química , Indóis/farmacologia , Testes de Sensibilidade Microbiana , Mutação , Oxidiazóis/química , Oxidiazóis/farmacologia , Nucleosídeos de Purina/química , Nucleosídeos de Purina/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , RNA Polimerase Dependente de RNA/metabolismo , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/fisiologia , Timina/análogos & derivados , Timina/química , Timina/farmacologia , Proteínas Virais/genéticaRESUMO
The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.
Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Negativas/enzimologia , Fenilalanina-tRNA Ligase/metabolismo , Sítios de Ligação , Farmacorresistência Bacteriana , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/genética , Testes de Sensibilidade Microbiana , Modelos Moleculares , Fenilalanina-tRNA Ligase/química , Sulfonamidas/farmacologiaRESUMO
Inhibitors of 4'-phosphopantetheine adenylyltransferase (PPAT) were identified through high-throughput screening of the AstraZeneca compound library. One series, cycloalkyl pyrimidines, showed inhibition of PPAT isozymes from several species, with the most potent inhibition of enzymes from Gram-positive species. Mode-of-inhibition studies with Streptococcus pneumoniae and Staphylococcus aureus PPAT demonstrated representatives of this series to be reversible inhibitors competitive with phosphopantetheine and uncompetitive with ATP, binding to the enzyme-ATP complex. The potency of this series was optimized using structure-based design, and inhibition of cell growth of Gram-positive species was achieved. Mode-of-action studies, using generation of resistant mutants with targeted sequencing as well as constructs that overexpress PPAT, demonstrated that growth suppression was due to inhibition of PPAT. An effect on bacterial burden was demonstrated in mouse lung and thigh infection models, but further optimization of dosing requirements and compound properties is needed before these compounds can be considered for progress into clinical development. These studies validated PPAT as a novel target for antibacterial therapy.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Animais , Antibacterianos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ligação Competitiva , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/química , Feminino , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Camundongos , Modelos Moleculares , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Panteteína/análogos & derivados , Panteteína/química , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/microbiologia , Bibliotecas de Moléculas Pequenas/química , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Coxa da Perna/microbiologiaRESUMO
A series of inhibitors with a squaramide core was synthesized following its discovery in a high-throughput screen for novel inhibitors of a transcription-coupled translation assay using Escherichia coli S30 extracts. The inhibitors were inactive when the plasmid substrate was replaced with mRNA, suggesting they interfered with transcription. This was confirmed by their inhibition of purified E. coli RNA polymerase. The series had antimicrobial activity against efflux-negative strains of E. coli and Haemophilus influenzae. Like rifampin, the squaramides preferentially inhibited synthesis of RNA and protein over fatty acids, peptidoglycan, and DNA. However, squaramide-resistant mutants were not cross-resistant to rifampin. Nine different mutations were found in parts of rpoB or rpoC that together encode the so-called switch region of RNA polymerase. This is the binding site of the natural antibiotics myxopyronin, corallopyronin, and ripostatin and the drug fidaxomicin. Computational modeling using the X-ray crystal structure of the myxopyronin-bound RNA polymerase of Thermus thermophilus suggests a binding mode of these inhibitors that is consistent with the resistance mutations. The squaramides are the first reported non-natural-product-related, rapidly diversifiable antibacterial inhibitors acting via the switch region of RNA polymerase.
Assuntos
Antibacterianos/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/metabolismo , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/enzimologia , Antibacterianos/química , RNA Polimerases Dirigidas por DNA/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Farmacorresistência Bacteriana , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Ensaios de Triagem em Larga Escala/métodos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/genética , Mutação , Ligação Proteica , Conformação Proteica , Rifampina/metabolismoRESUMO
The relationship between enzyme inhibition and antimicrobial potency of adenine-based NAD(+)-dependent DNA ligase (LigA) inhibitors was investigated using a strain of the Gram-negative pathogen Haemophilus influenzae lacking its major AcrAB-TolC efflux pump and the Gram-positive pathogen Streptococcus pneumoniae. To this end, biochemical inhibitors not mediating their antibacterial mode of action (MOA) via LigA were removed from the analysis. In doing so, a significant number of compounds were identified that acted via inhibition of LigA in S. pneumoniae but not in H. influenzae, despite being inhibitors of both isozymes. Deviations from the line correlating antimicrobial and biochemical potencies of LigA inhibitors with the correct MOA were observed for both species. These deviations, usually corresponding to higher MIC/IC50 ratios, were attributed to varying compound permeance into the cell.
RESUMO
N-Acylated ornithine analogues of daptomycin were synthesized and tested for their antibacterial efficacy.
Assuntos
Antibacterianos/síntese química , Daptomicina/análogos & derivados , Daptomicina/síntese química , Ornitina/análogos & derivados , Sepse/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológico , Animais , Antibacterianos/farmacologia , Daptomicina/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Ornitina/síntese química , Ornitina/farmacologia , Staphylococcus aureus/patogenicidadeRESUMO
Synthetic array technology was utilized to rapidly synthesize and analyze a diverse set of reductive alkylation analogues of daptomycin. Analysis of the array suggested the use of polar functionality such as sulfonamides or amide or polar spaces such as piperazine would beneficially affect activity.
Assuntos
Antibacterianos/síntese química , Daptomicina/análogos & derivados , Daptomicina/síntese química , Lipídeos/química , Oxirredução , Peptídeos Cíclicos/síntese química , Alquilação , Amidas/química , Antibacterianos/farmacologia , Técnicas de Química Combinatória , Daptomicina/farmacologia , Desenho de Fármacos , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecium/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Peptídeos Cíclicos/farmacologia , Piperazina , Piperazinas/química , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
Previous studies have suggested that lipoteichoic acid biosynthesis inhibition is the mechanism of action of daptomycin. In this investigation, daptomycin inhibited all macromolecular synthesis in Staphylococcus aureus, Enterococcus faecalis, and Enterococcus hirae without kinetic or dose specificity for lipoteichoic acid. Daptomycin remained bactericidal in the absence of ongoing lipoteichoic acid synthesis. Inhibition of lipoteichoic acid synthesis is apparently not the mechanism of action of daptomycin in these pathogens.