RESUMEN
The increasing emergence of antibiotic resistance necessitates the development of anti-infectives with novel modes of action. Targeting bacterial virulence is considered a promising approach to develop novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive antivirulence target. Mercaptoacetamide-based thiols have been reported to inhibit LasB as well as collagenases from clostridia and bacillus species. The present work provides an insight into the structure-activity relationship (SAR) of these fragment-like LasB inhibitors, demonstrating an inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An X-ray cocrystal structure is presented, revealing distinct binding of two compounds to the active site of LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human matrix metalloproteinases (MMPs).
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/química , Pseudomonas aeruginosa/efectos de los fármacos , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Animales , Antibacterianos/síntesis química , Sitios de Unión , Línea Celular , Cromatografía Liquida , Evaluación Preclínica de Medicamentos , Farmacorresistencia Bacteriana , Humanos , Espectrometría de Masas , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Mariposas Nocturnas/microbiología , Unión Proteica , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/síntesis química , Factores de VirulenciaRESUMEN
AIM: CsrA is a global post-transcriptional regulator protein affecting mRNA translation and/or stability. Widespread among bacteria, it is essential for their full virulence and thus represents a promising anti-infective drug target. Therefore, we aimed at the discovery of CsrA-RNA interaction inhibitors. Results & methodology: We followed two strategies: a screening of small molecules (A) and an RNA ligand-based approach (B). Using surface plasmon resonance-based binding and fluorescence polarization-based competition assays, (A) yielded seven small-molecule inhibitors, among them MM14 (IC50 of 4 µM). (B) resulted in RNA-based inhibitor GGARNA (IC50 of 113 µM). CONCLUSION: The first small-molecule inhibitors of the CsrA-RNA interaction were discovered exhibiting micromolar affinities. These hits represent tools to investigate the effects of CsrA-RNA interaction inhibition on bacterial virulence.