RESUMO
A series of dual targeting inhibitors of bacterial gyrase B and topoisomerase IV were identified and optimized to mid-to-low nanomolar potency against a variety of bacteria. However, in spite of seemingly adequate exposure achieved upon IV administration, the in vivo efficacy of the early lead compounds was limited by high levels of binding to serum proteins. To overcome this limitation, targeted serum shift prediction models were generated for each subclass of interest and were applied to the design of prospective analogs. As a result, numerous compounds with comparable antibacterial potency and reduced protein binding were generated. These efforts culminated in the synthesis of compound 10, a potent inhibitor with low serum shift that demonstrated greatly improved in vivo efficacy in two distinct rat infection models.
Assuntos
Antibacterianos/sangue , Bactérias/enzimologia , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , Inibidores da Topoisomerase II/sangue , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/enzimologia , Infecções Bacterianas/microbiologia , Proteínas Sanguíneas/metabolismo , DNA Topoisomerase IV/metabolismo , Humanos , Ratos , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/metabolismo , Inibidores da Topoisomerase II/farmacologiaRESUMO
High-throughput phenotypic screening against the yeast Saccharomyces cerevisiae revealed a series of triazolopyrimidine-sulfonamide compounds with broad-spectrum antifungal activity, no significant cytotoxicity, and low protein binding. To elucidate the target of this series, we have applied a chemogenomic profiling approach using the S. cerevisiae deletion collection. All compounds of the series yielded highly similar profiles that suggested acetolactate synthase (Ilv2p, which catalyzes the first common step in branched-chain amino acid biosynthesis) as a possible target. The high correlation with profiles of known Ilv2p inhibitors like chlorimuron-ethyl provided further evidence for a similar mechanism of action. Genome-wide mutagenesis in S. cerevisiae identified 13 resistant clones with 3 different mutations in the catalytic subunit of acetolactate synthase that also conferred cross-resistance to established Ilv2p inhibitors. Mapping of the mutations into the published Ilv2p crystal structure outlined the chlorimuron-ethyl binding cavity, and it was possible to dock the triazolopyrimidine-sulfonamide compound into this pocket in silico. However, fungal growth inhibition could be bypassed through supplementation with exogenous branched-chain amino acids or by the addition of serum to the medium in all of the fungal organisms tested except for Aspergillus fumigatus. Thus, these data support the identification of the triazolopyrimidine-sulfonamide compounds as inhibitors of acetolactate synthase but suggest that targeting may be compromised due to the possibility of nutrient bypass in vivo.
Assuntos
Acetolactato Sintase/antagonistas & inibidores , Antifúngicos/farmacologia , Pirimidinas/farmacologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Sulfonamidas/farmacologia , Compostos de Sulfonilureia/farmacologia , Acetolactato Sintase/química , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Aminoácidos de Cadeia Ramificada/farmacologia , Antifúngicos/química , Domínio Catalítico/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Pirimidinas/química , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Soro/química , Soro/metabolismo , Sulfonamidas/química , Compostos de Sulfonilureia/químicaRESUMO
Drug-resistant Gram-negative bacteria are of increasing concern worldwide. Novel antibiotics are needed, but their development is complicated by the requirement to simultaneously optimize molecules for target affinity and cellular potency, which can result in divergent structure-activity relationships (SARs). These challenges were exemplified during our attempts to optimize inhibitors of the bacterial enzyme CoaD originally identified through a biochemical screen. To facilitate lead optimization, we developed mass spectroscopy assays based on the hypothesis that levels of CoA metabolites would reflect the cellular enzymatic activity of CoaD. Using these methods, we were able to monitor the effects of cellular enzyme inhibition at compound concentrations up to 100-fold below the minimum inhibitory concentration (MIC), a common metric of growth inhibition. Furthermore, we generated a panel of efflux pump mutants to dissect the susceptibility of a representative CoaD inhibitor to efflux. These approaches allowed for a nuanced understanding of the permeability and efflux liabilities of the series and helped guide optimization efforts to achieve measurable MICs against wild-type E. coli.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Metabolômica/métodos , Nucleotidiltransferases/antagonistas & inibidores , Antibacterianos/síntese química , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Relação Estrutura-AtividadeRESUMO
In the preceding manuscript [ Moreau et al. 2018 , 10.1021/acs.jmedchem.7b01691 ] we described a successful fragment-based lead discovery (FBLD) strategy for discovery of bacterial phosphopantetheine adenylyltransferase inhibitors (PPAT, CoaD). Following several rounds of optimization two promising lead compounds were identified: triazolopyrimidinone 3 and 4-azabenzimidazole 4. Here we disclose our efforts to further optimize these two leads for on-target potency and Gram-negative cellular activity. Enabled by a robust X-ray crystallography system, our structure-based inhibitor design approach delivered compounds with biochemical potencies 4-5 orders of magnitude greater than their respective fragment starting points. Additional optimization was guided by observations on bacterial permeability and physicochemical properties, which ultimately led to the identification of PPAT inhibitors with cellular activity against wild-type E. coli.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/metabolismo , Benzimidazóis/síntese química , Benzimidazóis/química , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Sítios de Ligação , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mutação , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Ligação Proteica , Pirimidinonas/síntese química , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Triazóis/síntese química , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Compostos Heterocíclicos com 2 Anéis/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/metabolismo , Benzimidazóis/síntese química , Benzimidazóis/química , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Sítios de Ligação , Descoberta de Drogas , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Compostos Heterocíclicos com 2 Anéis/síntese química , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Ligação Proteica , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Pirimidinonas/síntese química , Pirimidinonas/química , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Triazóis/síntese química , Triazóis/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
The discovery of new antibacterial agents with novel mechanisms of action is necessary to overcome the problem of bacterial resistance that affects all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV are well-characterized clinically validated targets of the fluoroquinolone antibiotics which exert their antibacterial activity through inhibition of the catalytic subunits. Inhibition of these targets through interaction with their ATP sites has been less clinically successful. The discovery and characterization of a new class of low molecular weight, synthetic inhibitors of gyrase and topoisomerase IV that bind to the ATP sites are presented. The benzimidazole ureas are dual targeting inhibitors of both enzymes and possess potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. The discovery and optimization of this novel class of antibacterials by the use of structure-guided design, modeling, and structure-activity relationships are described. Data are presented for enzyme inhibition, antibacterial activity, and in vivo efficacy by oral and intravenous administration in two rodent infection models.