RESUMEN
Fragment-based screening (FBS) has gained acceptance in the pharmaceutical industry as an attractive approach for the identification of new chemical starting points for drug discovery programs in addition to classical strategies such as high-throughput screening. There is the concern that screening of fragments at high µM concentrations in biochemical assays results in increased false-positive and false-negative rates. Here the authors systematically compare the data quality of FBS obtained by enzyme activity-based fluorescence intensity, fluorescence lifetime, and mobility shift assays with the data quality from surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) methods. The serine protease trypsin and the matrix metalloprotease MMP12 were selected as model systems. For both studies, 352 fragments were selected each. From the data generated, all 3 biochemical protease assay methods can be used for screening of fragments with low false-negative and low false-positive rates, comparable to those achieved with the SPR-based assays. It can also be concluded that only fragments with a solubility higher than the screening concentration determined by means of NMR should be used for FBS purposes. Extrapolated to 10,000 fragments, the biochemical assays speed up the primary FBS process by approximately a factor of 10 and reduce the protease consumption by approximately 10,000-fold compared to NMR protein observation experiments.
Asunto(s)
Bioensayo/métodos , Evaluación Preclínica de Medicamentos/métodos , Metaloproteinasa 12 de la Matriz/metabolismo , Fragmentos de Péptidos/análisis , Tripsina/metabolismo , Animales , Bovinos , Cromatografía Liquida , Reacciones Falso Negativas , Reacciones Falso Positivas , Estudios de Factibilidad , Fluorescencia , Humanos , Cinética , Luz , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Fragmentos de Péptidos/química , Dispersión de Radiación , Solubilidad , Resonancia por Plasmón de SuperficieRESUMEN
To facilitate NMR spectroscopy studies of interactions with various ligands and potential inhibitors, we report the NMR backbone resonance assignments for the 26 kD human enzyme UCH-L3, a member of the ubiquitin C-hydrolase family of ubiquitin-specific cysteine proteases.
Asunto(s)
Cisteína Endopeptidasas/química , Dominio Catalítico , Cisteína Endopeptidasas/genética , Humanos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Ubiquitina TiolesterasaRESUMEN
Deubiquitinating proteases reverse protein ubiquitination and rescue their target proteins from destruction by the proteasome. USP2, a cysteine protease and a member of the ubiquitin specific protease family, is overexpressed in prostate cancer and stabilizes fatty acid synthase, which has been associated with the malignancy of some aggressive prostate cancers. Here, we report the structure of the human USP2 catalytic domain in complex with ubiquitin. Ubiquitin uses two major sites for the interaction with the protease. Both sites are required simultaneously, as shown by USP2 inhibition assays with peptides and ubiquitin mutants. In addition, a layer of ordered water molecules mediates key interactions between ubiquitin and USP2. As several of those molecules are found at identical positions in the previously solved USP7/ubiquitin-aldehyde complex structure, we suggest a general mechanism of water-mediated ubiquitin recognition by USPs.
