The prevalence of clinically-relevant bacterial strains resistant to current antibiotic therapies is increasing and has been recognized as a major health threat. For example, multidrug-resistant tuberculosis and methicillin-resistant Staphylococcus aureus are of global concern. Novel methodologies are needed to identify new targets or novel compounds unaffected by pre-existing resistance mechanisms. Recently, water-in-oil picodroplets have been used as an alternative to conventional high-throughput methods, especially for phenotypic screening. Here we demonstrate a novel microfluidic-based picodroplet platform which enables high-throughput assessment and isolation of antibiotic-resistant bacteria in a label-free manner. As a proof-of-concept, the system was used to isolate fusidic acid-resistant mutants and estimate the frequency of resistance among a population of Escherichia coli (strain HS151). This approach can be used for rapid screening of rare antibiotic-resistant mutants to help identify novel compound/target pairs.

The regional and cellular localization of the two subtypes of dopamine receptors, DI and D2, have been ascertained in rat forebrain by use of fluorescent dopaminergic antagonist ligands. (R,S)-5-(4'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-[1lH-3-benzazepin-7-ol, the 4'-amino derivative of the high-affinity Dl-specific antagonist SCH 23390, and the D2 selective antagonist N-(p-aminophenethyl)spiperone were chemically derivatized using the fluorescent compound tetramethylrhodamine. Two distinct populations of dopamine receptors exist in the nervous system (1). The D1 dopamine receptor subtype is positively linked to the activation of the cyclic 3',5'-adenosine monophosphate (cyclic AMP) second messenger system, while the D2 dopamine receptor subtype is linked to a variety of signal transduction mechanisms (2, 3). The pharmacological profiles of these two dopaminergic receptor populations can be discriminated on the basis of their agonist/antagonist specificities (4). The regional distributions of these receptor subtypes have been anatomically distinguished through in vitro autoradiographic localization of radiolabeled antagonist ligands. These previous studies indicate that both receptor subtypes are predominantly located in the basal ganglia and mesolimbic systems (5-7). Cellular localization of the striatal D1 dopamine receptor subtype can be approximated when radioligand binding is combined with the localization of the second messenger, cyclic AMP (8,9). Previous investigations have shown that the cyclic AMPreactive neurons in the caudate nucleus partially project to the substantia nigra (10), and biochemical investigations substantiate that the striatonigral neurons contain a dopamine-sensitive adenylate cyclase activity (11, 12) consistent with the presence of D1 dopamine receptor binding sites. Although the cellular localization of the striatal D2 receptor is unknown, it has been suggested that the cholinergic interneurons of the caudate nucleus have a D2 binding site (13). The recent report ofthe cDNA sequence for the rat brain D2 receptor may help to resolve this issue (14). Another approach taken by some of us (15) has been the specific derivatization of antagonists for the two dopamine receptor classes by using fluorescent moieties. The advantage of application of these derivatized D1-and D2-selective antagonist probes for anatomical determination of the dopamine receptor subtypes is that cellular localization of the binding sites can be distinguished with much higher resolution than by autoradiographic exposure techniques at these magnifications. We report here the regional and cellular localization of the D1 and D2 receptors in fresh-frozen tissue sections of intact rat forebrain using rhodamine-derivatized (R, S)-5-(4'-aminophenyl)-8-chloro-2,3 ,4,5-tetrahydro-3-methyl-[1Hl-3-benzazepin-7-ol (the 4'-amino derivative of SCH 23390) and N-(p-aminophenethyl)spiperone (NAPS) for the D1 and D2 receptors, respectively. METHODSMale Sprague-Dawley rats (200-250 g) were used to provide experim...

Aurora kinases are required for orderly progression of cells through mitosis. Inhibition of these kinases by siRNA or a small molecule inhibitors results in aberrant endoreduplication and cell death. SCH 1473759 is a novel Aurora inhibitor with potent mechanism based cell activity. The compound is active against a large panel of tumor cell lines from different tissue origin and genetic backgrounds. We found that asynchronous cells require 24 hour exposure to SCH 1473759 to induce maximal endoreduplication and cell kill. However, following a taxane or KSP inhibitor mitotic arrest, less than 4-hour exposure was sufficient to induce endoreduplication. This finding correlated with the ability of SCH 1473759 to accelerate exit from mitosis in response to taxane and KSP induced arrest, but not that of a nocodazole arrest. SCH 1473759 demonstrated single agent biomarker and anti-tumor activity in A2780 ovarian xenograft models. Further, efficacy was enhanced in combination with taxotere and found to be most efficacious when SCH 1473759 was dosed 12-hours post taxotere. These findings could have clinical implications for the development of Aurora inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1648.

Agonist shift assays feature cross-titrations of allosteric modulators and orthosteric ligands. Information generated in agonist shift assays can include a modulator's effect on the orthosteric agonist's potency (alpha) and efficacy (beta), as well as direct agonist activity of the allosteric ligand (tauB) and the intrinsic binding affinity of the modulator to the unoccupied receptor (KB). Because of the heavy resource demand and complex data handling, these allosteric parameters are determined infrequently during the course of a drug discovery program and on a relatively small subset of compounds. Automation of agonist shift assays enables this data-rich analysis to evaluate a larger number of compounds, offering the potential to differentiate compound classes earlier and prospectively prioritize based on desired molecular pharmacology. A high-throughput calcium-imaging agonist shift assay was pursued to determine the allosteric parameters of over 1000 positive allosteric modulator (PAM) molecules for the human muscarinic acetylcholine receptor 1 (M). Control compounds were run repeatedly to demonstrate internal consistency. Comparisons between potency measurements and the allosteric parameter results demonstrate that these different types of measurements do not necessarily correlate, highlighting the importance of fully characterizing and understanding the allosteric properties of leads.