CCHTS Contents and Abstracts, Vol 3, No. 1

Volume 3, Number 1, 2000: Contents

Screening for Genetic Mutations. A Review. Pp. 1-9.
Masato Tawata, Kaoru Aida and Toshimasa Onaya
[Abstract]

High-Throughput Screening of Genetic Mutations/Polymorphisms by Capillary Electrophoresis. Pp. 11-25.
Jicun Ren
[Abstract]

Measurement of Cdk4 Kinase Activity Using an Affinity Peptide-Tagging Technology. Pp. 27-36.
Jinzi J. Wu, Donna R. Yarwood, Bhabatosh Chaudhuri, Lionel Muller, Mauro Zurini and Matthew A. Sills
[Abstract]

Solid-Phase Synthesis and Biological Screening of N-a-Mercaptoamide Template-Based Matrix Metalloprotease Inhibitors. Pp. 37-41.
J.F. Lynas, S.L. Martin, B. Walker, A.D. Baxter, J. Bird, R. Bhogal, J.G. Montana and D.A. Owen
[Abstract]

A Solution-Phase Combinatorial Synthesis of Selective Dopamine D₄ Ligands. Pp. 43-50.
John P. Williams and Karine Lavrador
[Abstract]

Generation of a Polyclonal Fab Phage Display Library to the Human Breast Carcinoma Cell Line BT-20. Pp. 51-57.
K. E. Santora, S. Sarantopoulos, W. Den, S. Petersen-Mahrt, S. R. Sompuram, and J. Sharon
[Abstract]

Abstracts

[Back to top] Screening for Genetic Mutations. A Review. Masato Tawata, Kaoru Aida and Toshimasa Onaya.
A point mutation of a nucleotide within a single gene can have a profound effect on a specific organ and/or the entire human body. DNA sequences associated with human diseases may differ from the corresponding normal sequences by single nucleotide mutations or by large alterations such as deletions, insertions, duplications, or translocations of DNA segments or entire chromosomes. As a result of the heterogeneity of DNA alterations and genetic mutations, various screening approaches are required to detect these alterations. However, methods which facilitate the detection of large alterations in the genome are typically insensitive to point mutations, whereas methods which detect point mutations are not appropriate to detect large alterations within the genome. Since there is no single perfect method to screen for unknown mutations, combinations of these methods may be necessary for accurate genetic diagnosis. The applications of polymerase chain reaction (PCR) technology to genomic screening have made rapid and accurate genetical diagnosis possible. Furthermore, recent developments in the technology of DNA microarrays have opened the way for high throughput sequence analysis by hybridization, which shows great potential in both molecular biology and medicine in the near future.

[Back to top] High-Throughput Screening of Genetic Mutations/Polymorphisms by Capillary Electrophoresis. Jicun Ren.
Genetic mutations/polymorphisms analyses play a great role in genetic and medical research, and clinical diagnosis. Most conventional methods for genetic assay are based on slab gel electrophoresis that is both labor-intensive and time-consuming. Recently, capillary electrophoresis (CE) has been used for genetic analysis instead of conventional slab gel electrophoresis. This technique can be automated and is characterized by short analysis time, small sample and reagents requirements, and high separation efficiency. CE has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes, and has shown a great potential for genetic mutation/polymorphism screening of large numbers of clinical samples. In this article, an overview of the fundamental aspects of mutation/polymorphism assay methods in combination with CE is given and some key applications are summarized.

[Back to top] Measurement of Cdk4 Kinase Activity Using an Affinity Peptide-Tagging Technology. Jinzi J. Wu, Donna R. Yarwood, Bhabatosh Chaudhuri, Lionel Muller, Mauro Zurini and Matthew A. Sills.
: Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.

[Back to top] Solid-Phase Synthesis and Biological Screening of N-a-Mercaptoamide Template-Based Matrix Metalloprotease Inhibitors. .J.F. Lynas, S.L. Martin, B. Walker, A.D. Baxter, J. Bird, R. Bhogal, J.G. Montana and D.A. Owen
A series of N-a-mercaptoacetyl containing dipeptides have been prepared on solid-phase supports as putative matrix metalloprotease (MMP) inhibitors. Inhibitor design was based on a positional scanning approach of the amino acids present within a template molecule, previously shown to be an MMP inhibitor with good pharmacological characteristics. This study is the first step in a unique programme, designed to expand the repertoire of molecular templates which can be chosen as starting points for the development of more focused parallel and/or combinatorial libraries of MMP inhibitors as a means to accelerate the lead discovery process. This paper reports the success of such an approach in the development of agents with activity against a number of pathologically important MMPs. After screening of these positional scanning libraries, we have obtained important SAR information, in particular, pharmacophores with the ability to impart selectivity for particular MMP species.

[Back to top] A Solution-Phase Combinatorial Synthesis of Selective Dopamine D₄ Ligands. John P. Williams and Karine Lavrador.
Utilizing combinatorial synthesis and a preparative LC-MS automated chromatography system we have prepared and purified a library of 4-[2-(1,2,4-oxadiazolyl)]piperidines that were designed to be novel and selective dopamine D₄ ligands. In one round of synthesis we identified N-4-chlorobenzyl-4-[2-(3-(2-thienyl)-1,2,4-oxadiazolyl)]piperidine with a K_d of 5 nM for the human D₄ receptor.

[Back to top] Generation of a Polyclonal Fab Phage Display Library to the Human Breast Carcinoma Cell Line BT-20. K. E. Santora, S. Sarantopoulos, W. Den, S. Petersen-Mahrt, S. R. Sompuram, and J. Sharon.
We have previously described a vector system for generating recombinant polyclonal antibody libraries. This system uses bidirectional phagemid and mammalian expression vectors to facilitate mass transfer of selected variable light and variable heavy (VL-VH) region gene pairs from the phagemid to the mammalian vector, to express polyclonal libraries of whole IgG antibodies. We report here the first stage of generating a polyclonal antibody library to the human breast carcinoma cell line BT-20, using this vector system. VL and VH region gene pairs were obtained from a mouse immunized with BT-20 cells, and cloned, in opposite transcriptional orientations, in the bidirectional phagemid vector, to produce an Fab phage display library. This library was selected by panning on BT-20 cells and shown to bind specifically to BT-20 cells. Such libraries, after suitable negative selection to eliminate major reactivities against normal tissue, could be transferred in mass to our bidirectional mammalian expression vector for production of libraries of chimeric antibodies with mouse V regions and human constant (C) regions. These polyclonal antibody libraries will mediate effector functions and are expected to be useful for breast cancer therapy, as well as diagnosis.