Contents
Molecular
Recognition: Design of “Keys” Pp.409-427
Beining Chen, Sergey Piletsky and Anthony P.F. Turner
High-Density
Synthetic Peptide Microarrays: Emerging Tools for Functional Genomics and
Proteomics Pp.429-440
Ronald
Frank
Enhancement
of Combinatorial Chemistry by Microwave-Assisted Organic Synthesis Pp.441-458
Pelle
Lidström, Jacob Westman and Anthony Lewis
Improvement
of “Hit-to-Lead” Optimization by Integration of in Vitro HTS Experimental
Models for Early Determination of Pharmacokinetic Properties Pp.459-472
Ilona
Kariv, Robyn A. Rourick, Daniel B. Kassel and Thomas D.Y. Chung
Wheat
Germ Cell-Free Translation System as a Tool for In vitro Selection of
Functional Proteins Pp.473-480
A.N.
Alexandrov, V. Yu. Alakhov and A.I. Miroshnikov
Solution-Phase
Parallel Synthesis of an Isoflavone Library for the Discovery of Novel
Antigiardial Agents Pp.481-487
T.
Mineno, K.M. Stanford, L.A. Walker and M.A. Avery
Generation
of Anti-Colorectal Cancer Fab Phage Display Libraries With a High Percentage of
Diverse Antigen-Reactive Clones
Pp.489-499
Brent
R. Williams, Seshi R. Sompuram and Jacqueline Sharon
High
Throughput Synthesis of Ester Library Utilizing Selective Molecular Reactivity
and Recognition Technology
Pp.501-502
Atul
Kumar, Sangita and S. Ray
[Back to top] Molecular Recognition: Design of “Keys”
Beining
Chen, Sergey Piletsky and Anthony P.F. Turner
Molecular recognition between
molecules is one of the most fundamental processes in biology and chemistry.
The recognition process is largely driven by non-covalent forces such as
hydrogen bonding, electrostatics, van der Waals forces, p-p
interactions, and
conformational energy. The complementarity between the receptor and substrate
is very similar to the "lock and key" function, first described by
Emil Fischer over 100 years ago, - the lock being the molecular receptor such
as a protein or enzyme and the key being the substrate such as a drug, that is
recognized to give a defined receptor-substrate complex. This review focuses on
the design of specific ligand systems as “Keys” to enable the induced fit of
these keys into the target macromolecules, protein/enzyme (Locks) with
particular emphasis on protein recognition.
[Back to top] High-Density Synthetic Peptide
Microarrays: Emerging Tools for Functional Genomics and Proteomics
Ronald
Frank
New approaches for manufacturing and application of
peptide arrays on planar surfaces are emerging, thereby opening advanced
opportunities to probe the expression and function of the proteome. In
complementing DNA and protein array analyses, peptide fragment screening
directly addresses functional protein interaction sites, leading to a detailed
insight into the discovered molecular recognition events, placing them in the
context of the whole genome, and even allowing rapid determination of the
chemical nature of these interactions. This information can then be transferred
into powerful small peptide tools that interfere with these interactions in
vivo and help to link targets with phenotypes. With the spreading of new
peptide array tools, peptide screening will extend its impact on modern
genome-driven molecular biology. This will advance the systematic discovery and
validation of new pharmaceutical targets as well as the development of potent
molecular diagnostics for medical and ecological monitoring.
[Back to top] Enhancement
of Combinatorial Chemistry by Microwave-Assisted Organic Synthesis
Pelle
Lidström, Jacob Westman and Anthony Lewis
It was in the 1980’s that the first papers in which the
use of either combinatorial methods or microwave heating in organic chemistry
were published. Unlike combinatorial chemistry, which quite readily became an
accepted method, particularly in the pharmaceutical industry, it is only now
that microwave heating is truly gaining acceptance. Our aim in this review is
to attempt to rationalize this slow acceptance and to show the benefits to be
gained by employing microwave heating in tandem with combinatorial chemistry.
We will also give a number of examples of successful applications.
[Back to top] Improvement
of “Hit-to-Lead” Optimization by Integration of in Vitro HTS Experimental
Models for Early Determination of Pharmacokinetic Properties
Ilona Kariv, Robyn A. Rourick, Daniel B. Kassel and Thomas D.Y. Chung
Development of predictive in vitro surrogate methods for traditional
approaches assessing bioavailability and pharmacokinetics of lead compounds
must be made to both keep pace with highthroughput (HT) lead identification and
to mitigate the high costs associated with progression of compounds with poor
chances of developmental success. Indeed opportunities for improvement still
exist in the lead optimization phase versus the lead identification phase,
where HT methodologies have been nearly optimized. Review of examples,
limitations, and development of high-throughput microtiterplate-based assays
for evaluating metabolic liabilities, such as in vitro radiometric and
fluorometric assays for inhibition of cytochrome P450 (CYP) activity,
determination of stability of a compound in liver microsomes, or cloned CYPs coupled
to reconstituting systems are described. Parallel approaches to improve speed,
resolution, sample preparation, as well as data analysis using LC/MS and
LC/MS/MS approaches and technologies to assess compound integrity and
biotransformation by automation and multiplexing are also discussed.
Realization of the benefits in automation of cell-based models for determining
drug permeability to predict drug absorption are still hampered by bottlenecks
in analytical analysis of compounds. The implementation and limitations of
surrogate physiochemical methods for passive adsorption such as immobilized
artificial membranes (IAM) and parallel artificial membrane permeation assays
(PAMPA), and compound solubility by laser nephelometry are reviewed as well.
Additionally, data from a high-throughput 96-well equilibrium dialysis device,
showing good correlation to classical methods, is presented. Finally, the
impact of improvements in these downstream bottlenecks in lead optimization and
preclinical drug discovery are discussed in this review.
[Back to top] Wheat Germ Cell-Free Translation System as a
Tool for In vitro Selection of Functional Proteins
A.N. Alexandrov, V. Yu. Alakhov and A.I. Miroshnikov
We have demonstrated that mRNA, ribosome and resulting
protein form complexes (ternary complexes) in wheat germ cell-free translation
system and these complexes are stable for at least several hours. The protein
folds into a proper conformation capable of specific binding with the inhibitor
of its enzymatic activity. The removal of the stop codon from mRNA does not
affect translation and mRNA-ribosome-protein complex stability. We have used
these results to develop a method of isolation of mouse dihydrofolate reductase
(mDHFR) encoding mRNA from native pool of mouse liver mRNA. The native pool of
mouse liver mRNA was translated in vitro in a wheat germ cell-free translation
system (WG-CFS), and enzyme-specific ternary complexes were affinity selected
on a methotrexate-BSA coated 96-well microtiter plate (methotrexate, MTX, is an
inhibitor of DHFR enzymatic activity). Bounded ternary complexes were eluted by
MTX treatment. mRNA from eluates was amplified by template-switch RT-PCR and
products of RT-PCR analyzed by gel electrophoresis. The cDNA was amplified by
one-step reverse transcription-PCR and used for transcription, followed by
translation and determination of the DHFR enzymatic activity in translation
mixtures. This method is suitable for direct cDNA cloning from mRNA or cDNA
libraries and for investigation of protein-protein interactions.
[Back to top] Solution-Phase Parallel Synthesis of an Isoflavone
Library for the Discovery of Novel Antigiardial Agents
T. Mineno, K.M. Stanford, L.A. Walker and M.A. Avery
Combinatorial chemistry has become a dramatically useful
tool for the development of new medicinal agents. In the search to discover a
novel and effective lead for the treatment of giardiasis, solutionphase
synthesis of a library of isoflavone derivatives has been accomplished. Of the
products screened, several compounds such as P(A1,B1) and P(A1,B11) exhibited
potent antigiardial activity. The details of synthesis, in vitro antigiardial
assay, and preliminary structure-activity relationships of these compounds are
described.
[Back to top] Generation
of Anti-Colorectal Cancer Fab Phage Display Libraries With a High Percentage of
Diverse Antigen-Reactive Clones
Brent
R. Williams, Seshi R. Sompuram and Jacqueline Sharon
A combinatorial Fab phage display library was generated
from the antibody variable region genes of each of 2 BALB/c mice immunized with
the human colorectal cancer cell lines SW480, SW948, and SW837. These libraries
were shown to be diverse by nucleotide sequencing and diagnostic restriction
enzyme digestion (fingerprinting) of individual members. The two libraries were
combined and selected for binding to a suspension of formaldehyde-fixed human
colorectal cancer cells in two successive rounds of selection and phage
amplification by infection of bacteria. Analysis of the selected libraries as
well as individual library clones by ELISA, showed binding to the cancer cell
lines in both formaldehyde-fixed and native forms. Fifty five percent and 94%
of library clones were positive for colorectal cancer cell binding after the
first and second rounds of selection, respectively. Fingerprinting of
individual clones showed the first round selected library to be very diverse
and the second round selected library to be of more limited diversity. After
absorption with normal human cell types, these anti-cancer selected libraries
could be used to develop therapeutic and/or diagnostic agents.
[Back to top] High Throughput Synthesis of Ester
Library Utilizing Selective Molecular Reactivity and Recognition Technology
Atul Kumar, Sangita and S. Ray
This paper reports a new solid support reagent that showed
high degrees of selective molecular reactivity and molecular recognition in
homo-functional reactions (reactions having similar reactive functionality in
reactants and products).