Combinatorial Chemistry & High Throughput Screening - Volume 7, Issue 1, 2004

As 2003 begins, Combinatorial Chemistry & High Throughput Screening has completed its six year of publication. During the last year, several new members joined the Editorial Board replacing others whose years of service were much appreciated. In particular, I would like to acknowledge the addition of the following new members of our Editorial Board: Dr. Brian Kay, Argonne National Laboratories, Argonne, IL. Brian has already published papers in CCHTS in the area of phage display, and another paper from his groups appears in this issue. Since we receive so many manuscripts in the areas of epitope mapping, biopanning, and phage display, his expertise is most appreciated. Dr. David Goodlett, University of Washington, Seattle, WA. David is an expert in proteomics, systems biology and mass spectrometry. We expect to receive more papers in this area during the next few years, and his help will be invaluable. Dr. Gunda Georg, University of Kansas, Lawrence, KS. Gunda has already published on combinatorial synthesis in CCHTS and will help us with the review of manuscripts in this area. Dr. Matthew Kerby, Palo Alto, CA. Matt honed his knowledge of microfluidics and chip technology while working at Caliper Technologies. During the next few years, the number of manuscripts in this expanding field should increase. Dr. J. Michael Ramsey, Oak Ridge National Laboratory, TN. Mike will help us review papers on nanotechnology, microseparations, and mass spectrometry. During 2003, Combinatorial Chemistry & High Throughput Screening will continue to publish review articles and original research papers in all areas of combinatorial chemistry and high throughput screening. For example, articles in this and forthcoming issues will address developments in combinatorial synthesis and high throughput screening as well as their applications. For example, the manuscript by Kämpke discusses structure retrieval in chemical data bases, which is an essential component for the design of structurally diverse combinatorial libraries. The paper by Han, et al., describes an approach to accelerate the screening of phage-displayed libraries for affinity reagents, and the review by Gerner addresses the use of proteomics for the identification of diagnostic markers. Heilker et al. describe the use of time-resolved fluorescence and fluorescence polarization for high throughput screening, and Chen and Ren review the use of microchip capillary electrophoresis for high throughput DNA analysis including analysis of PCR products, DNA fragments, and DNA mutations. Combinatorial Chemistry & High Throughput Screening is abstracted and indexed by the major services including BIOSIS, Chemical Abstracts, Current Contents / Life Sciences, EMBASE, BIOBASE, Science Citation Index-Expanded, Index Medicus / MEDLINE, and CAB Abstracts. As a result, articles published in Combinatorial Chemistry & High Throughput Screening are highly visible to the research community. Once again, we plan to publish eight issues of CCHTS during 2004, and this frequency of publication remains the highest in the field of combinatorial chemistry or high throughput screening. 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. Authors will be pleased to learn that we accept manuscripts in either paper or electronic format, and our readers and subscribers will continue be able to obtain Combinatorial Chemistry & High Throughput Screening in printed or electronic format. The field of combinatorial chemistry continues to grow in importance as does the associated area of high throughput screening. The union of these interdependent disciplines in a single journal including both review articles and original research papers makes Combinatorial Chemistry & High Throughput Screening a unique and essential scientific journal. Once again I thank the distinguished members of our Editorial Board, our Regional Editors, our able Guest Editors, the authors who contributed reviews and research papers, and of course you, our readers, for the success and continuing growth of our journal.

Drugs exert their functions mainly by affecting proteins. Therefore, it seems straightforward to focus on proteins in order to investigate drug effects. Unfortunately, proteins are of very high complexity, rendering it much more difficult to screen for protein alterations as compared to gene regulation. However, the efficiencyand applicability of proteome analysis has been dramatically increased recently. We are on the way to be ableto comprehensively assess disease-related proteome alterations, which may become an essential source ofinformation for knowledge-based drug design. This review will provide an overview of current techniques inproteome analysis, focusing on screening technologies for biomedical research. An outlook at the futurepotential of proteomics supported by modern bioinformatics will highlight why proteomics is worth the effort.

Screening a library of molecular graphs for an exact or approximate match with one particular molecular graph, the query graph, is reduced to list comparisons. The lists contain lengths of shortest paths ingraph Voronoi regions. This induces the notion of shortest path similarity. All graphs that are shortest path similar to the query graph are efficiently retrievable. The same applies to approximate or similarity matching. For the retrieval of all superstructures of a query, shortest path lists are modified to distance patterns. This alsoallows algorithmic support for query construction.

We introduce a graphical representation of DNA primary sequences by taking four special vectors in a 3-D space to represent the four nucleic acid bases in DNA sequences, so that a DNA primary sequence isdenoted in a 3-D space by a successive vector sequence which is a directed walk on the space. It is demonstrated that this representation has no overlap and intersection and allows numericalcharacterization.

DNA analysis plays a great role in genetic and medical research, and clinical diagnosis of inherited diseases and particular cancers. Development of new methods for high throughput DNA analysis is necessitated with incoming of post human genome era. A new powerful analytical technology, called microchip capillary electrophoresis (MCE), can be integrated with some experimental units and is characterized by high-speed, small sample and reagent requirements and high-throughput. This new technology, which has been applied successfully to the separation of DNA fragments, analysis of polymerasechain reaction (PCR) products, DNA sequencing, and mutation detection, for example, will become anattractive alternative to conventional methods such as slab gel electrophoresis, Southern blotting andNorthern blotting for DNA analysis. This review is focused on some basic issues about DNA analysis by MCE,such as fabrication methods for microchips, detection system and separation schemes, and several keyapplications are summarized.

Time-resolved fluorescence (TRF) assay formats are frequently used technologies in highthroughput screening. In this article, we have characterised the novel Plate::Vision2 96-microlens array reader (Carl Zeiss Jena GmbH, Germany) and compared it to the novel LEADseeker Generation IV multimodality imaging system (LEADseeker Gen IV;; Amersham Biosciences UK Ltd., UK) for applications in the TRF mode. In europium measurements using the TRF mode, the Plate::Vision displayed a limit of detection for europium of approximately 3 pM, which was comparable to two established TRF readers, the Discovery and the Victor V (both PerkinElmer Life Sciences Inc., USA). The LEADseeker's limit of detection only extended down to europium concentrations of approximately 10 pM in these experiments. For TRF resonance energy transfer (TR-FRET) experiments, a europium-biotin (Eu-biotin) conjugate was titrated with a streptavidin-allophycocyanin (SA-APC) conjugate. The Plate::Vision produced Z' values larger than 0.5 for the acceptor fluorophor emission with concentrations of Eu-biotin as low as 3 nM combined with 175 pM SA-APC. To achieve Z' values of at least 0.5 with the LEADseeker, concentrations of 10 nM Eu-biotin combined with SA-APC of at least 0.8 nM were required. In a drug screening application using TR-FRET, the energy transfer from a europium-labelled protein X (Eu-protein X) to a complex of biotinylated peptide Y with SA-APC was measured. Using the Plate::Vision, a Z' factor larger than 0.5 for the acceptor fluorophor emission was only obtained for a Eu-protein X concentration of at least 10 nM in combination with biotinylated peptide Y / /SA-APC at saturating concentrations. Both the Plate::Vision and the LEADseeker show good quality results for applications in the TRF mode and enable an increased throughput based on their shortened measurement time in comparison to classic photomultiplier tube-based readers.

When using multiple targets and libraries, selection of affinity reagents from phage-displayed libraries is a relatively time-consuming process. Herein, we describe an automation-amenable approach to accelerate the process by using alkaline phosphatase (AP) fusion proteins in place of the phage ELISA screening and subsequent confirmation steps with purified protein. After two or three rounds of affinity selection, the open reading frames that encode the affinity selected molecules (i.e., antibody fragments, engineered scaffold proteins, combinatorial peptides) are amplified from the phage or phagemid DNA molecules by PCR and cloned en masse by a Ligation Independent Cloning (LIC) method into a plasmid encoding a highly active variant of E. coli AP. This time-saving process identifies affinity reagents that work out of context of the phage and that can be used in various downstream enzyme linked binding assays. The utility of this approach was demonstrated by analyzing single-chain antibodies (scFvs), engineered fibronectin type III domains (FN3), and combinatorial peptides that were selected for binding to the Epsin Nterminal Homology (ENTH) domain of epsin 1, the c-Src SH3 domain, and the appendage domain of the gamma subunit of the clathrin adaptor complex, AP-1, respectively.

In this paper we introduce a quantitative model that relates chemical structural similarity to biological activity, and in particular to the activity of lead series of compounds in high-throughput assays. From this model we derive the optimal screening collection make up for a given fixed size of screening collection, and identify the conditions under which a diverse collection of compounds or a collection focusing on particular regions of chemical space are appropriate strategies. We derive from the model a diversity function that may be used to assess compounds for acquisition or libraries for combinatorial synthesis by their ability to complement an existing screening collection. The diversity function is linked directly through the model to the goal of more frequent discovery of lead series from high-throughput screening. We show how the model may also be used to derive relationships between collection size and probabilities of lead discovery in high-throughput screening, and to guide the judicious application of structural filters.