Combinatorial Chemistry & High
Throughput Screening, Vol. 6, No. 2, 2003
Contents
Metal-Enhanced
Fluorescence: Potential Applications in HTS Pp.109-117
Chris D. Geddes , Ignacy Gryczynski , Joanna Malicka , Zygmunt Gryczynski and Joseph R. Lakowicz
Potential
of Peptides Selected from Random Phage-Displayed Libraries to Mimic
Conformational Epitopes: A Study on Scorpion Toxin Cn2 and the Neutralizing
Monoclonal Antibody BCF2 Pp.119-132
Tatiana
G. Gazarian , Barbara Selisko , Georgina B. Gurrola , Ricardo Hernández ,
Lourival D. Possani and Karlen G.
Gazarian
Traceless
Liquid Phase Synthesis of Piperazinediones Pp.133-137
Chung-Ming
Sun , K.M.K Swamy, Mei-Jung Lin, Wen-Ben Yeh, Fred Y. Chen and Wei-Hong Tseng
Tetraacyldiborates:
Selective and Efficient Acylation Reagents Suitable for Multiple Parallel
Synthetic Applications Pp.139-145
Elmer
J. Gentry, Hanumaiah Telikepalli, Pusuluri Srinivas and Lester A. Mitscher
Rapid
Identification of Allergen-Encoding cDNA Clones by Phage Display and
High-Density Arrays Pp.147-154
Rimantas
Kodzius , Claudio Rhyner , Zoltan Konthur , Donald Buczek , Hans Lehrach ,
Gerald Walter and Reto Crameri
Inhibition
of Hepatitis C Virus Serine Protease in Living Cells by RNA Aptamers Detected
Using Fluorescent Protein Substrates Pp.155-160
N.
Kakiuchi1, K. Fukuda , F. Nishikawa ,
S. Nishikawa and K. Shimotohno
SAR
Modeling: Effect of Experimental Ambiguity Pp.161-166
Bhavani
P. Thampatty and Herbert S. Rosenkranz
Abstracts
[Back to top] Metal-Enhanced
Fluorescence: Potential Applications in HTS
Chris
D. Geddes , Ignacy Gryczynski , Joanna Malicka , Zygmunt Gryczynski and Joseph R. Lakowicz
Metallic surfaces and particles can have dramatic effects on fluorescence, including localized excitation, increased quantum yields, increased photostability and increased distances for resonance energy transfer (RET), and directional emission. While all these effects have not yet been realized in a single system, metal-enhanced fluorescence promises to provide the next generation of high sensitivity fluorescence assays for low copy number detection of biochemical species.
[Back to top] Potential of Peptides Selected from
Random Phage-Displayed Libraries to Mimic Conformational Epitopes: A Study on
Scorpion Toxin Cn2 and the Neutralizing Monoclonal Antibody BCF2
Tatiana
G. Gazarian , Barbara Selisko , Georgina B. Gurrola , Ricardo Hernández ,
Lourival D. Possani and Karlen G.
Gazarian
Many conformational epitopes cannot be mapped by the use of a phage display approach due to the lack of amino acid similarity with the selected peptides. Exploring the potential of the method, we selected mimotopes of the discontinuous, highly conformational epitope of scorpion neurotoxin Cn2, whose 3D structure is known, using its generic neutralizing monoclonal antibody BCF2. With an exhaustive selection procedure, we isolated from a 12-mer phage library a large collection of mimotopes that reproduce the antigenic and immunogenic specificity of the Cn2-epitope. The selected peptides presented three sequence motifs, the most abundant of which, RD(N)XXGF, appeared in 15 different sequence contexts displayed by 97 out of 206 clones. In the most reactive mimotope, displayed by 24 (25%) clones, the motif was flanked by two Cys residues allowing the adoption of a cyclic conformation. Motifs QL(H,M)L(M) and (S/T)WHLP were\ selected with less efficiency. Comparison of the motifs with the primary and three-dimensional structure of Cn2 as well as with a model of the Cn2-BCF2(Fv) complex suggests that RD(N)XXGF, which does not share sequence similarity with the epitope, mimics its central structural element, turn 7-11, by using an alternative amino acid combination nevertheless keeping the nature of its interactions with BCF2. The QL(H,M)L(M) is assumed to mimic the hydrophobic part of the epitope. The principles of the conformational mimicry by phage-displayed peptides are discussed.
[Back to top] Traceless
Liquid Phase Synthesis of Piperazinediones
Chung-Ming
Sun , K.M.K Swamy, Mei-Jung Lin, Wen-Ben Yeh, Fred Y. Chen and Wei-Hong Tseng
Liquid phase combinatorial synthesis (LPCS) of piperazinediones by the use of soluble polymer support is explored to generate libraries. Proline anchored polyethylene glycol monomethyl ether (PEG) underwent dipeptide formation with different Fmoc-amino acids in the presence of dicyclohexyl carbodiimide (DCC). Deprotection of Fmoc accompanied with the cleavage from polymer support with cyclization of dipeptide to piperazinediones offers a facile and effective way to prepare diverse combinatorial libraries. Excellent yields and purities were achieved by simple wash and precipitation method.
[Back to top] Tetraacyldiborates: Selective and Efficient Acylation Reagents Suitable
for Multiple Parallel Synthetic Applications
Elmer J. Gentry, Hanumaiah Telikepalli, Pusuluri Srinivas and Lester A. Mitscher
Boron-based mixed anhydrides are rapidly reactive, easy to prepare, cheap, efficient, and general acylating reagents capable of selectivity when chelation is possible. High yields of various esters, amides and thioesters are quickly obtainable and the products are easy to isolate in high purity. The method is readily used under multiple parallel synthesis conditions and is readily scaleable.
[Back to top] Rapid Identification of
Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays
Rimantas Kodzius , Claudio Rhyner , Zoltan Konthur , Donald Buczek , Hans Lehrach , Gerald Walter and Reto Crameri
We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries. The strategy is based on a combination of phage display and high-density arrays. To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinityselected on solid phase-immobilized serum IgE from allergic patients. Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking. CDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes. Filters were iteratively probed with randomly-sequenced inserts until all clones were identified. Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found. This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries.
[Back to top] Inhibition of Hepatitis C
Virus Serine Protease in Living Cells by RNA Aptamers Detected Using
Fluorescent Protein Substrates
N. Kakiuchi1, K. Fukuda , F. Nishikawa , S. Nishikawa and K. Shimotohno
Hepatitis C virus is one of the causative agents of non-A non-B hepatitis. Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation. NS3 serine protease is considered to be a suitable target for anti-HCV reagents. We report an assay of HCV NS3 protease in living cells. We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP. Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease. The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed. As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection. We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro. We studied the effect of RNA aptamers introduced into HeLa cells. The addition of G9-II RNA in the medium at a concentration of 2.5 µg/ml reduced cleavage by onethird that of control.
[Back to top] SAR Modeling: Effect of
Experimental Ambiguity
Bhavani P. Thampatty and Herbert S. Rosenkranz
The applicability of SAR
(structure-activity relationship) techniques to data obtained using high
throughput screening (HTS) and toxicogenomic techniques is explored. The reason
for this study derives from the fact that for economical and time
considerations HTS bioassays may consist of single determinations, i.e. lack of
duplication. This introduces an element of uncertainty. Using two different
data bases of fairly complex biological phenomena (allergic contact dermatitis
in humans and the induction of mutations in Salmonella), it is demonstrated
that the resulting SAR models can tolerate up to 20% ambiguity in the
experimental data.