A Merger of Rational Drug Design and Combinatorial Chemistry: Development and Application of Peptide Secondary Structure Mimetics. Pp. 167-183.
Recombinant Polyclonal Antibody Libraries. Pp. 185-196.
J. Sharon*, S.
Sarantopoulos, W. Den, C.-Y. Kao, C. M. Baecher-Allan, K. E. Santora, S. R.
Sompuram, S. Petersen-Mahrt and B. R. Williams
Redistribution in Combinatorial Synthesis. A Theoretical Approach. Pp. 197-209.
Árpád Furka*
PCR/SSCP Detects Reliably and Efficiently DNA Sequence Variations in Large Scale Screening Projects. Pp. 211-218.
Bianca Miterski, Rejko Krüger,
Philip Wintermeyer and Jörg T. Epplen*
Rational Drug Design and High-Throughput Techniques for RNA Targets. Pp. 219-234.
Thomas Hermann* and Eric Westhof
Identification of Differential Gene Expression by High Throughput Analysis. Pp. 235-241.
Kin-Ying To*
Nucleic Acids
for Recognition and Catalysis: Landmarks, Limitations, and Looking to the
Future. Pp. 243-269.
David M. Perrin*
[Back to top] A Merger of Rational Drug Design and Combinatorial Chemistry: Development and Application of Peptide Secondary Structure Mimetics.
Hwa-Ok Kim* and Michael Kahn
Recent efforts toward the development of peptide secondary structure mimetics at Molecumetics Ltd. for the discoveries of new drug candidates utilizing combinatorial chemistry with solid phase synthesis are described.
[Back to top] Recombinant Polyclonal Antibody Libraries.
J. Sharon*, S. Sarantopoulos, W. Den, C.-Y. Kao, C. M. Baecher-Allan, K. E. Santora, S. R. Sompuram, S. Petersen-Mahrt and B. R. Williams
We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen). Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen 'escape variants', and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations. The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries. This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies. We review here our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.
[Back to top] Redistribution in Combinatorial Synthesis. A Theoretical Approach.
Árpád Furka*
In modified versions of the split-mix method, the conventional solid support resin was replaced by labeled macroscopic support units in order to produce individual compounds in multi milligram quantities while the high productivity of the original procedure is preserved. Recently it has also been shown that tagging the units is unnecessary. Omission of the tags is possible if (i) the unlabeled support units are arranged into spatially ordered groups and the relative spatial arrangement of the units is maintained during the chemical reactions (ii) the support units are redistributed between the reaction steps according to a predetermined pattern permitted by the combinatorial redistribution rule and (iii) the sorting process is simulated by a computer that can trace the synthetic history of each support unit. Different kinds of solid support units, formation of spatially ordered groups, sorting devices and basic redistribution patterns (serial, semi-parallel and parallel) are discussed. It is also shown that particularly the semi-parallel and the parallel redistribution assure fast sorting.
[Back to top] PCR/SSCP Detects Reliably and Efficiently DNA Sequence Variations in Large Scale Screening Projects.
Bianca Miterski, Rejko Krüger, Philip Wintermeyer and Jörg T. Epplen*
A simple and fast method with high reliability is necessary for the identification of mutations, polymorphisms and sequence variants (MPSV) within many genes and many samples, e.g. for clarifying the genetic background of individuals with multifactorial diseases. Here we review our experience with the polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) analysis to identify MPSV in a number of genes thought to be involved in the pathogenesis of multifactorial neurological disorders, including autoimmune diseases like multiple sclerosis (MS) and neurodegenerative disorders like ParkinsonĆs disease (PD). The method is based on the property of the DNA that the electrophoretic mobility of single stranded nucleic acids depends not only on their size but also on their sequence. The target sequences were amplified, digested into fragments ranging from 50-240 base pairs (bp), heat-denatured and analysed on native polyacrylamide (PAA) gels of different composition. The analysis of a great number of different PCR products demonstrates that the detection rate of MPSV depends on the fragment lengths, the temperature during electrophoresis and the composition of the gel. In general, the detection of MPSV is neither influenced by their location within the DNA fragment nor by the type of substitution, i.e., transitions or transversions. The standard PCR/SSCP system described here provides high reliability and detection rates. It allows the efficient analysis of a large number of DNA samples and many different genes.
[Back to top] Rational Drug Design and High-Throughput Techniques for RNA Targets.
Thomas Hermann* and Eric Westhof
RNA molecules are the only known molecules which possess the double property of being depository of genetic information, like DNA, and of displaying catalytic activities, like protein enzymes. RNA molecules intervene in all steps of gene expression and in many other biological activities. Like proteins, RNAs achieve those biological functions by adopting intricate three-dimensional folds and architectures. Further, as in protein sequences, RNA sequences contain signatures specific for three-dimensional motifs which participate in recognition and binding. In regulatory pathways, RNA molecules exist in equilibria between transient structures differentially stabilized by effectors such as proteins or cofactors. Therefore, RNA molecules display their potential as drug targets on different levels, namely in three-dimensional folds, in structural equilibria and in RNA-protein interfaces. Several examples will be described together with the already available techniques for combinatorial synthesis and high-throughput screening of potential drug and target RNA molecules.
[Back to top] Identification of Differential Gene Expression by High Throughput Analysis.
Kin-Ying To*
High throughput analysis of differential gene expression is a powerful tool that can be applied to many areas in molecular cell biology, including differentiation, development, physiology, and pharmacology. In recent years, a variety of techniques have been developed to analyze differential gene expression, including comparative expressed sequence tag sequencing, differential display, representational difference analysis, cDNA or oligonucleotide arrays, and serial analysis of gene expression. This review explains the technologies, their scopes, impact on science, as well as their costs and possible limitations. The application of differential display is presented as a tool to identify genes induced by darkness or yellowing process in rice leaves.
[Back to top]
Nucleic Acids for Recognition and Catalysis:
Landmarks, Limitations, and Looking to the Future.
David M. Perrin*
Combinatorial selection of nucleic acids has led to the discovery of novel ligands and catalysts that have implications for both chemistry and medicine. In the context of combinatorial chemistry, degenerate syntheses of nucleic acid libraries readily generate as many as 1015 different molecules in which a small percentage exhibit interesting binding and/or catalytic properties. The primary advantage of nucleic acids is that library coding is an intrinsic property; sequential composition directly determines the activity. At low temperatures, the sequential composition of single stranded nucleic acids governs folding into irregular tertiary structures resulting in interesting activities. At higher temperatures, the same structures are unfolded and decoded by polymerases to reveal sequential information. The use of PCR (polymerase chain reaction) permits amplification and thus enrichment of the selected activity which is then regenerated chemi-enzymatically. Iterative selection and amplification result in one of the highest throughput screens conceivable whereby each molecule encodes its own activity permitting the ultimate in parallel sampling. Finally, sequence information, and by extension the chemical composition, is obtained by simple sequencing techniques obviating the need for mass spectrometric deconvolution, parallel tagging, and/or large volumes needed for viral and cell culture. This review begins with an introduction of general concepts and considerations. The potential for nucleic acids to generate tight-binding ligands is of interest to structural biologists and medicinal chemists. The therapeutic implications to medicine are also touched upon. Since combinatorially selected nucleic acids and antibodies share many conceptual similarities, their respective advantages and limitations are compared. Theoretical and practical limitations for catalyst discovery are discussed along with the use of other chemical and physical approaches to address some current catalytic shortcomings. Finally some future directions are suggested.