RESUMO
Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5' tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5' leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.
Assuntos
Antraquinonas/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Ribonuclease P/antagonistas & inibidores , Antraquinonas/química , Antraquinonas/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Corantes Fluorescentes , Fluorometria , Hematoxilina/análogos & derivados , Hematoxilina/química , Hematoxilina/metabolismo , Hematoxilina/farmacologia , Simulação de Dinâmica Molecular , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Ribonuclease P/química , Ribonuclease P/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
One of the hallmarks of life is the widespread use of certain essential ribozymes. The ubiquitous ribonuclease P (RNase P) and eukaryotic RNase MRP are essential complexes where a structured, noncoding RNA acts in catalysis. Recent discoveries have elucidated the three-dimensional structure of the ancestral ribonucleoprotein complex, suggested the possibility of a protein-only composition in organelles, and even noted the absence of RNase P in a non-free-living organism. With respect to these last two findings, import mechanisms for RNases P/MRP into mitochondria have been demonstrated, and RNase P is present in organisms with some of the smallest known genomes. Together, these results have led to an ongoing debate regarding the precise definition of how "essential" these ribozymes truly are.