No abstract

Caspase-3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase-3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase-3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e. CFP-DEVD-YFP). We demonstrate that, once initiated, the activation of caspase-3 is a very rapid process, taking 5 min or less to reach completion. Furthermore, this process occurs almost simultaneously with a depolarization of the mitochondrial membrane potential. These events occur just prior to the characteristic morphological changes associated with apoptosis. Our results clearly demonstrate that, once initiated, the commitment of cells to apoptosis is a remarkably rapid event when visualized at the single cell level.

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

No abstract

]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed ␦-opioid receptors and ␤ 2 -adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the ␦-opioid receptor-␤ 2 -adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human ␦-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.Recent studies have started to provide a significant body of evidence to support a concept of constitutive homo-oligomerization of a range of G protein-coupled receptors (GPCRs) 1 (1, 2).GPCRs for which such evidence exists include the ␤ 2 -adrenoreceptor (3, 4), the D 2 dopamine receptor (5), the M 3 muscarinic acetylcholine receptor (6), the V 2 vasopressin receptor (7), the ␦-opioid (8, 9) and -opioid receptors (9), the histamine H 2 receptor (10), and the CCR5 receptor (11). Furthermore, recent evidence has also indicated a requirement for the constitutive hetero-oligomerization of distinct GPCRs, such as between the GABA B R1 and GABA B R2 receptors, to generate a functional receptor expressed at the cell surface (12). Two important issues, however, remain contentious. The first of these is whether ligand occupancy alters the extent of GPCR oligomerization, and the second is the likely extent of GPCR hetero-oligomerization. In a range of reports, GPCR homo-oligomerization has been reported to be increased (3, 4, 11), decreased (8), or unaffected (6, 7) by the addition of receptor ligands. Similarly, in GPCR heterodimerization studies, interactions have been indicated to be unaffected (12), regulated (13), or almost entirely dependent upon (14) the addition of receptor agonists and hetero-oligomers have recently been reported to form between quite distinct (14), as well as between closely related (12, 13), GPCR sequences. The earliest studies on GPCR oligomerization relied on the capacity to co-immunoprecipitate co-expressed but differentially epitope-tagged forms of a GPCR (see Ref. 15 for review). Because of the hydrophobic nature of the seven trans-plasma membrane helices of GPCR family members, care must be taken, however, to exclude nonspecific interactions between GPCR pairs resulting from detergent dissolution of cellular membranes. More recent studies have employed vari...

Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics. Compound 11 demonstrated in vivo efficacy in an S. aureus rat abscess infection model.

No abstract

The thrust of early drug discovery in recent years has been toward the configuration of homogeneous miniaturized assays. This has allowed organizations to contain costs in the face of exponential increases in the number of screening assays that need to be run to remain competitive. Miniaturization brings with it an increasing dependence on instrumentation, which over the past several years has seen the development of nanodispensing capability and sophisticated detection strategies. To maintain confidence in the data generated from miniaturized assays, it is critical to ensure that both compounds and reagents have been delivered as expected to the target wells. The authors have developed a standard operating procedure for liquid-handling quality control that has enabled them to evaluate performance on 2 levels. The first level provides for routine daily testing on existing instrumentation, and the second allows for more rigorous testing of new dispensing technologies. The procedure has shown itself to be useful in identifying both method programming and instrumentation performance shortcomings and has provided a means to harmonizing instrumentation usage by assay development and screening groups. The goal is that this type of procedure be used for facilitating the exchange of liquid handler performance data across the industry.