RESUMEN
The global rise of metallo-ß-lactamases (MBLs) is problematic due to their ability to inactivate most ß-lactam antibiotics. MBL inhibitors that could be coadministered with and restore the efficacy of ß-lactams are highly sought after. In this study, we employ virtual screening of candidate MBL inhibitors without thiols or carboxylates to avoid off-target effects using the Avalanche software package, followed by experimental validation of the selected compounds. As target enzymes, we chose the clinically relevant B1 MBLs NDM-1, IMP-1, and VIM-2. Among 32 compounds selected from an approximately 1.5 million compound library, 6 exhibited IC50 values less than 40 µM against NDM-1 and/or IMP-1. The most potent inhibitors of NDM-1, IMP-1, and VIM-2 had IC50 values of 19 ± 2, 14 ± 1, and 50 ± 20 µM, respectively. While chemically diverse, the most potent inhibitors all contain combinations of hydroxyl, ketone, ester, amide, or sulfonyl groups. Docking studies suggest that these electron-dense moieties are involved in Zn(II) coordination and interaction with protein residues. These novel scaffolds could serve as the basis for further development of MBL inhibitors. A procedure for renaming NDM-1 residues to conform to the class B ß-lactamase (BBL) numbering scheme is also included.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/clasificación , beta-Lactamasas/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Dicroismo Circular , Simulación por Computador , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Enzimológica de la Expresión Génica , Espectrometría de Masas , Modelos Químicos , Estructura Molecular , Programas InformáticosRESUMEN
Metallo-ß-lactamases inactivate most ß-lactam antibacterials, and much attention has been paid to their catalytic mechanism. One issue of controversy has been whether ß-lactam hydrolysis generally proceeds through an anionic intermediate bound to the active-site Zn(II) ions or not. The formation of an intermediate has not been shown conclusively in imipenemase (IMP) enzymes to date. Here, we provide evidence that intermediates are formed during the hydrolysis of meropenem and chromacef catalyzed by the variant IMP-25 and, to a lesser degree, IMP-1.
Asunto(s)
Antibacterianos/metabolismo , Cefalosporinas/metabolismo , Tienamicinas/metabolismo , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Catálisis , Dominio Catalítico , Hidrólisis , Cinética , Meropenem , Zinc/metabolismoRESUMEN
How eukaryotic ribosomes traverse messenger RNA (mRNA) leader sequences to search for protein-synthesis start sites remains one of the most mysterious aspects of translation and its regulation. While the search process is conventionally described by a linear "scanning" model, its exquisitely dynamic nature has restricted detailed mechanistic study. Here, we observed single Saccharomyces cerevisiae ribosomal scanning complexes in real time, finding that they scan diverse mRNA leaders at a rate of 10 to 20 nt s-1. We show that specific binding of a protein to its mRNA leader sequence substantially arrests scanning. Conversely, impairing scanning-complex guanosine 5'-triphosphate hydrolysis results in native start-site bypass. Our results illustrate an mRNA-centric, kinetically controlled regulatory model where the ribosomal pre-initiation complex amplifies a nuanced energetic landscape to regulate scanning and start-site selection fidelity.