Combinatorial Chemistry & High
Throughput Screening, Vol. 7, No. 1, 2004
Contents
Screening
for Disease-Markers and Investigating Drug Effects by Proteome Profiling: Can
it Meet Expectations? Pp.1-9
Christopher Gerner
Scalable
Distance Similarity of Chemical Structures Pp.11-21
Thomas
Kampke
On
a 3-D Representation of DNA Primary Sequences Pp.23-27
Chun
Li and Jun Wang
High-Throughput
DNA Analysis by Microchip Electrophoresis Pp.29-43
Lin
Chen and Jicun Ren
Time-Resolved
Fluorescence Measurements Using Microlens Array and Area Imaging Devices
Pp.45-54
Susanne
Merk, Achim Lietz, Margareta Kroner, Martin Valler and Ralf Heilker
Accelerated
Screening of Phage-Display Output with Alkaline Phosphatase Fusions
Pp.55-62
Zhaozhong
Han, Ece Karatan, Michael D. Scholle, John McCafferty and Brian K. Kay
Design
of a Compound Screening Collection for use in High Throughput Screening
Pp.63-70
G.
Harper, S.D. Pickett and D.V.S. Green
Abstracts
[Back to top] Screening
for Disease-Markers and Investigating Drug Effects by Proteome Profiling: Can
it Meet Expectations?
Christopher
Gerner
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 efficiency and applicability of proteome analysis has been dramatically increased recently. We are on the way to be able to comprehensively assess disease-related proteome alterations, which may become an essential source of information for knowledge-based drug design. This review will provide an overview of current techniques in proteome analysis, focusing on screening technologies for biomedical research. An outlook at the future potential of proteomics supported by modern bioinformatics will highlight why proteomics is worth the effort.
[Back to top] Scalable Distance Similarity of
Chemical Structures
Thomas
Kampke
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 in graph 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 also allows algorithmic support for query construction.
[Back to top] On a 3-D
Representation of DNA Primary Sequences
Chun
Li and Jun Wang
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 is denoted 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 numerical characterization.
[Back to top] High-Throughput DNA Analysis by Microchip Electrophoresis
Lin Chen and Jicun Ren
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 polymerase chain reaction (PCR) products, DNA sequencing, and mutation detection, for example, will become an attractive alternative to conventional methods such as slab gel electrophoresis, Southern blotting and Northern 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 key applications are summarized.
[Back to top] Time-Resolved Fluorescence Measurements Using Microlens Array
and Area Imaging Devices
Susanne Merk, Achim Lietz, Margareta Kroner, Martin Valler and Ralf Heilker
Time-resolved fluorescence (TRF)
assay formats are frequently used technologies in high-throughput 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
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.
[Back to top] Accelerated Screening of Phage-Display Output with Alkaline
Phosphatase Fusions
Zhaozhong Han, Ece Karatan, Michael D. Scholle, John McCafferty and Brian K. Kay
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 N-terminal 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.
[Back to top] Design of a Compound Screening Collection for use in High
Throughput Screening
G. Harper, S.D. Pickett and D.V.S. Green
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.