Combinatorial Chemistry & High Throughput Screening - Volume 9, Issue 1, 2006

With this issue Combinatorial Chemistry & High Throughput Screening begins its ninth year of publication. CCHTS continues to publish review articles and original research papers in all areas of combinatorial chemistry and high throughput screening. This issue, for example, contains both review articles (see the review by Hsieh, et al., of fast mass spectrometric assays that are being used to enhance the throughput of drug metabolism and pharmacokinetics studies) and research papers (see the paper by Allan, et al., describing the development of a new fluorescence-based HTS assay for inhibitors of glycine transporters). Synthetic organic chemistry papers featuring new routes to combinatorial libraries will continue to appear in CCHTS, such as the research paper by Coelho and Sotelo in this issue that describes palladium catalyzed multicomponent reactions leading to the synthesis of pyridazinone libraries. In addition, the development of new HTS assays will be covered; see for example the paper by Han, et al., describing a new peptide chip screening approach for caspase activity. As always, the use of screening assays for applicants such as drug discovery will continue to appear in CCHTS, such as the paper by Wood, et al., that concerns the use of HTS to discover anti-endotoxin agents. As in the past, regular issues will be alternated with special issues focusing on a single topic of current interest. Some of these special issues will be organized by members of our Editorial Board whereas others will be organized by guest editors. For example, the second issue of CCHTS to be published in 2006 has been organized by Dr. Donald Kyle, who is a member of the Editorial Board. Dr. Kyle's special issue will address how to increase the productivity of drug discovery programs, and topics to be covered will include library synthesis strategies based on computational predictions of biological activity, utilization of corporate databases, and comparison of approaches for sequential library screening. Whether appearing in a special issue addressing a hot topic or in a regular issue, all papers published in CCHTS will continue to be peer-reviewed. In 2005, eight issues of CCHTS were published, and this frequency of publication was the highest of any journal in the field of combinatorial chemistry or high throughput screening. Due to the large number of manuscripts in the publication queue, the number of issues of CCHTS planned for 2006 will be increased to ten. Therefore, CCHTS will set a new standard this year as it continues to be the publication leader in this field. Papers published in CCHTS are abstracted and indexed by the major services including Chemical Abstracts, BIOSIS, CAB Abstracts, EMBASE, BIOBASE, Science Citation Index-Expanded, MEDLINE, and Current Contents (Life Sciences). The homepage of our journal and abstracts of articles may be found at the following Internet address: http://www.bentham.org/cchts. Information for authors may also be found at our website. Manuscripts may be submitted in print or electronic format, and our readers and subscribers will continue to be able to obtain CCHTS in printed or electronic format. Through the combination of frequent publication and high visibility, Combinatorial Chemistry & High Throughput Screening remains a unique and essential scientific journal defining the intersection of these two interdependent disciplines. I would like to thank the distinguished members of the Editorial Board, the Regional Editors, the guest editors, the authors, and of course you, the readers, for the continuing success of our journal.

Historically, most bioanalytical methods for drug analysis in pharmaceutical industry were developed using HPLC coupled with UV or fluorescence detection. However, there is a trend toward interfacing separation technologies with more sensitive tandem mass spectrometry (MS/MS)-based systems. MS/MS detection offers complete resolution of the parent compounds from their first pass metabolites to avoid extra efforts for separation and sample clean-up procedures resulting in shorter run times. With the increasing demand for ever faster screening, there is a continuing demand for bioanalytical methods possessing higher sample throughput for both in vitro and in vivo drug metabolism and pharmacokinetic evaluations to accelerate the discovery process. This review focuses on the current approaches for fast MS-based assays (cycle-time less than 5 min) of pharmaceuticals and their metabolites that have been reported in the peer-reviewed publications.

The glycine transporter (GlyT-1b) is a Na+/Cl--dependent electrogenic transporter which mediates the rapid reuptake of glycine from the synaptic cleft. Based on its tissue distribution, GlyT-1 has been suggested to co-localise with the NMDA receptor where it may modulate the concentration of glycine at its co-agonist binding site. This data has led to GlyT-1 inhibitors being proposed as targets for disorders such as schizophrenia and cognitive dysfunction. Radiolabelled uptake assays (e.g. [3H]glycine) have been traditionally used in compound screening to identify glycine transporter inhibitors. While such an assay format is useful for testing limited numbers of compounds, the identification of novel glycine uptake inhibitors requires a functional assay compatible with high-throughput screening (HTS) of large compound libraries. Here, the authors present the development of a novel homogenous cell-based assay using the FLIPR membrane potential blue dye (Molecular Devices) and FLEXstation. Pharmacological data for the GlyT-1 inhibitors Org 24598 and ALX 5407 obtained using this novel electrogenic assay correlated well with the conventional [3H]-glycine uptake assay format. Furthermore, the assay has been successfully miniaturised using FLIPR3 and therefore has the potential to be used for high-throughput screening.

A simple and straightforward three-component methodology for the solution phase high-throughput synthesis of 2,5-disubstituted pyridazin-3(2H)-one libraries has been developed. The diversity at position 5 of the heterocycle is determined by the use of Suzuki, Sonogashira, Heck or Stille coupling.

Proteases play a key role in cell functions, and it is very important to monitor their activities for drug screening and diagnosis of diseases. In the present study, a new class of fluorescence probe, into which a fluorophore and a quencher have been introduced, was developed and applied to the on-chip detection of caspase-3 activity. This probe is non fluorescent in the absence of caspase-3. However, when it is treated with active caspase-3, the fluorescence intensity increases dependent on the caspase-3 activity due to the cleavage of the quencher-containing moiety on a glass slide. This caspase-dependent increase in the fluorescence intensity was also detected when the glass slide immobilizing the probe peptide was treated with cell lysate stimulated by staurosporine (STP), which is an apoptosis-inducing agent. On the other hand, such an increase was not detected in the case of control cell lysate without STP-stimulation. The developed system is a rapid and sensitive method and is useful for the direct measurement of protease activity on a glass array.

Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are an integral part of the outer leaflet of the outermembrane of Gram-negative bacteria. Lipopolysaccharides play a pivotal role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient, worldwide. The sequestration of circulatory endotoxin may be a viable therapeutic strategy for the prophylaxis and treatment of Gram-negative sepsis. We have earlier shown that the pharmacophore necessary for small molecules to bind LPS involves two protonatable cationic functions separated by about 15 Å, permitting the simultaneous interaction with the negatively charged phosphates on lipid A, the toxically active center of endotoxin. In this report, screening of a multi-thousand membered polyamine library through the combined use of computational and bioassay-guided screens resulted in the discovery of two novel classes of LPS-binding agents. These are represented by the 1) spermine sulfonamides and 2) C-aryl-substituted spermine analogs. We present the selection approach, screening results, computational multivariate analyses and initial structure-activity relationship evaluation herein.

High Content Screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modelling systems. In this work, we compared a confocal and a non-confocal cellular HCS system, the IN Cell Analyzers1 3000™ and 1000™, respectively. As a cellular model system we used the Transfluor™ 2 technology in the 384-well microtiter plate (MTP) format. The Transfluor HCS assay for G-protein coupled receptor (GPCR) activation is based on the recruitment of a green fluorescent protein-labelled arrestin (ArrGFP) from the cytosol to the plasma membrane. We investigated two GPCRs, the wild-type (wt) β2 adrenergic receptor ( β2AR) and the β2AR-enhanced (E), a C-terminally mutated receptor with a higher affinity to arrestin. Upon agonist stimulation, the β2AR-wt induced the redistribution of ArrGFP to coated pits, the β2AR-E maintained the interaction with ArrGFP down to the formation of endocytic vesicles. Our findings reveal that the assay is feasible on both instruments, with sufficiently robust Z' statistics. Improved Z' statistics, though, are achieved with the confocal system, particularly in case of weak signals. Moreover, throughput is dramatically higher for the IN Cell Analyzer 3000. We conclude that, depending on the needs for throughput and assay biology, either instrument may fulfil a successful role in the drug discovery process. Confocal optics, however, provide a better basis for the detection of smaller subcellular structures with lower fluorescence intensity.

In this paper, a new protein coding gene-finding algorithm specific for the yeast genome at 96% accuracy is proposed. Five-fold cross-validation tests are performed to confirm the above accuracy. By the new algorithm, the number of protein coding genes in the yeast genome is re-estimated. The estimate is based on the assumption that the unknown genes have similar statistical properties to the known genes. It is found that the number of protein coding genes in the 16 yeast chromosomes is less than or equal to 5873, significantly coincident with the widely accepted range 5800-6000.

Phage display is one of the best methods to identify drug targets, although technical problems including imprecision in quantifying phage and false-positive results are common. To address these difficulties, we propose two methods to more rapidly identify drug-binding phage particles. First, quantification of phage using SYBR Green Realtime PCR significantly improved accuracy and reproducibility. Second, affinity-column chromatography for selection of drug-binding phage particles concentrated particles more than a 96-well plate, making a phage amplification step, which can bias phage distribution, unnecessary. The methods proposed here should be suitable for high-throughput phagedisplay screenings and ultimately lead to more rapid identification of drug targets.

The oligosaccharide residue in glycoconjugates located in cell membranes is responsible for intercellular recognition and interaction: it acts as a receptor for proteins, hormones, and microorganisms and governs immune reactions. These significant activities have stimulated great interest in the field of oligosaccharides and glycoconjugates. Although many advances have been made in the synthesis of oligosaccharides, more convenient and efficient methods are still needed. This review describes one of these new methods-the one-pot sequential glycosylation approach as a potent tool for oligosaccharide assembly. The oligosaccharide library construction in a one-pot fashion is also summarized.