RESUMO
Previous work established a coumarin scaffold as a starting point for inhibition of Mycobacterium tuberculosis (Mtb) FadD32 enzymatic activity. After further profiling of the coumarin inhibitor 4 revealed chemical instability, we discovered that a quinoline ring circumvented this instability and had the advantage of offering additional substitution vectors to further optimize. Ensuing SAR studies gave rise to quinoline-2-carboxamides with potent anti-tubercular activity. Further optimization of ADME/PK properties culminated in 21b that exhibited compelling in vivo efficacy in a mouse model of Mtb infection.
Assuntos
Antituberculosos/química , Proteínas de Bactérias/antagonistas & inibidores , Cumarínicos/química , Animais , Antituberculosos/metabolismo , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Quinolinas/química , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated.