RESUMO
DNA gyrase is an essential enzyme in bacteria, and its inhibition results in the disruption of DNA synthesis and, subsequently, cell death. The pyrrolamides are a novel class of antibacterial agents targeting DNA gyrase. These compounds were identified by a fragment-based lead generation (FBLG) approach using nuclear magnetic resonance (NMR) screening to identify low-molecular-weight compounds that bind to the ATP pocket of DNA gyrase. A pyrrole hit with a binding constant of 1 mM formed the basis of the design and synthesis of a focused library of compounds that resulted in the rapid identification of a lead compound that inhibited DNA gyrase with a 50% inhibitory concentration (IC(50)) of 3 µM. The potency of the lead compound was further optimized by utilizing iterative X-ray crystallography to yield DNA gyrase inhibitors that also displayed antibacterial activity. Spontaneous mutants were isolated in Staphylococcus aureus by plating on agar plates containing pyrrolamide 4 at the MIC. The resistant variants displayed 4- to 8-fold-increased MIC values relative to the parent strain. DNA sequencing revealed two independent point mutations in the pyrrolamide binding region of the gyrB genes from these variants, supporting the hypothesis that the mode of action of these compounds was inhibition of DNA gyrase. Efficacy of a representative pyrrolamide was demonstrated against Streptococcus pneumoniae in a mouse lung infection model. These data demonstrate that the pyrrolamides are a novel class of DNA gyrase inhibitors with the potential to deliver future antibacterial agents targeting multiple clinical indications.
Assuntos
Amidas/farmacologia , Antibacterianos/farmacologia , Pirróis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Inibidores da Topoisomerase II , Amidas/química , Animais , Antibacterianos/química , Sítios de Ligação , Cristalografia por Raios X , DNA Girase/química , DNA Girase/metabolismo , Farmacorresistência Bacteriana , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Mutação , Ligação Proteica , Pirróis/química , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Streptococcus pneumoniae/crescimento & desenvolvimentoRESUMO
We present the discovery and optimization of a novel series of bacterial topoisomerase inhibitors. Starting from a virtual screening hit, activity was optimized through a combination of structure-based design and physical property optimization. Synthesis of fewer than a dozen compounds was required to achieve inhibition of the growth of methicillin-resistant Staphyloccus aureus (MRSA) at compound concentrations of 1.56 µM. These compounds simultaneously inhibit DNA gyrase and Topoisomerase IV at similar nanomolar concentrations, reducing the likelihood of the spontaneous occurrence of target-based mutations resulting in antibiotic resistance, an increasing threat in the treatment of serious infections.
Assuntos
Antibacterianos/química , DNA Topoisomerase IV/antagonistas & inibidores , Inibidores Enzimáticos/química , Indóis/química , Inibidores da Topoisomerase II , Adenosina Trifosfatases/química , Antibacterianos/síntese química , Antibacterianos/farmacologia , Compostos Aza/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Girase/metabolismo , DNA Topoisomerase IV/metabolismo , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Indóis/síntese química , Indóis/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
The pyrrolamides are a new class of antibacterial agents targeting DNA gyrase, an essential enzyme across bacterial species and inhibition results in the disruption of DNA synthesis and subsequently, cell death. The optimization of biochemical activity and other drug-like properties through substitutions to the pyrrole, piperidine, and heterocycle portions of the molecule resulted in pyrrolamides with improved cellular activity and in vivo efficacy.
Assuntos
Amidas/química , Antibacterianos/química , Antibacterianos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirróis/química , Inibidores da Topoisomerase II , Amidas/síntese química , Amidas/farmacologia , Antibacterianos/síntese química , Bactérias/efeitos dos fármacos , Sítios de Ligação , Cristalografia por Raios X , DNA Girase/metabolismo , Inibidores Enzimáticos/síntese química , Testes de Sensibilidade Microbiana , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
AZD5099 (compound 63) is an antibacterial agent that entered phase 1 clinical trials targeting infections caused by Gram-positive and fastidious Gram-negative bacteria. It was derived from previously reported pyrrolamide antibacterials and a fragment-based approach targeting the ATP binding site of bacterial type II topoisomerases. The program described herein varied a 3-piperidine substituent and incorporated 4-thiazole substituents that form a seven-membered ring intramolecular hydrogen bond with a 5-position carboxylic acid. Improved antibacterial activity and lower in vivo clearances were achieved. The lower clearances were attributed, in part, to reduced recognition by the multidrug resistant transporter Mrp2. Compound 63 showed notable efficacy in a mouse neutropenic Staphylococcus aureus infection model. Resistance frequency versus the drug was low, and reports of clinical resistance due to alteration of the target are few. Hence, 63 could offer a novel treatment for serious issues of resistance to currently used antibacterials.
Assuntos
Amidas/farmacologia , Antibacterianos/farmacologia , Pirróis/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Tiazóis/farmacologia , Inibidores da Topoisomerase II/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Amidas/síntese química , Amidas/química , Animais , Antibacterianos/síntese química , Antibacterianos/química , Cristalografia por Raios X , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Knockout , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Pirróis/síntese química , Pirróis/química , Ratos , Ratos Wistar , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/química , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/químicaRESUMO
The discovery and optimization of a new class of bacterial topoisomerase (DNA gyrase and topoisomerase IV) inhibitors binding in the ATP domain are described. A fragment molecule, 1-ethyl-3-(2-pyridyl)urea, provided sufficiently potent enzyme inhibition (32 µM) to prompt further analogue work. Acids and acid isosteres were incorporated at the 5-pyridyl position of this fragment, bridging to a key asparagine residue, improving enzyme inhibition, and leading to measurable antibacterial activity. A CF3-thiazole substituent at the 4-pyridyl position improved inhibitory potency due to a favorable lipophilic interaction. Promising antibacterial activity was seen versus the Gram-positive pathogens Staphylococcus aureus and Streptococcus pneumoniae and the Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis . Precursor metabolite incorporation and mutant analysis studies support the mode-of-action, blockage of DNA synthesis by dual target topoisomerase inhibition. Compound 35 was efficacious in a mouse S. aureus disease model, where a 4.5-log reduction in colony forming units versus control was demonstrated.