Phage Display in Proteolysis and Signal Transduction. Pp. 19-28.
Hermann Gram
[Abstract]
A Novel Cleavage Protocol For Use With Rf-Encoded Split Pool Synthesis
Technology: Product Cleavage and Collection in Standard 96 Well Format.
Pp. 29-32.
C. J. Andres, R. T. Swann, K. Grant-Young, Stanley V. D'Andrea and
Milind S. Deshpande
[Abstract]
A Solid Phase Approach to Substituted Pyrimidines and Their Conversion
into Condensed Heterocycles for Potential Use in Combinatorial Chemistry.
Pp. 33-38.
Sanjay K. Srivastava, W. Haq and P.M.S. Chauhan
[Abstract]
High Throughput Liquid Chromatography-Mass Spectrometry Assessment of
the Metabolic Activity of Commercially Available Hepatocytes from 96-Well
Plates. Pp. 39-52.
Gory W. Caldwell, John A. Mosucci and Enrique Chacon
[Abstract]
[Back to top] Phage Display
in Proteolysis and Signal Transduction. Hermann Gram.
The power of the phage display technology relies on the coupling of
the functional display of combinatorial peptide or protein libraries with
the ability of each member in the library to self-replicate and, at the
same time, to encode the primary structure of the displayed polypeptide
in its genome. Phage display systems, therefore, reflect the principle
of encoded combinatorial chemistry close to perfection. Phage display libraries
have extensively been used for the selection of peptides, antibody combining
sites or protein variants binding to given structures such as polypeptides,
carbohydrates, nucleic acids or small molecular weight compounds. The use
of peptide libraries in selecting molecular interaction partners was extensively
described in numerous publications and was subject to a variety of review
articles in the past. More recently, and in the focus of this review, combinatorial
phage libraries have been employed to examine substrate recognition in
catalysis and signal transduction. The sensitivity and versatility of phage
display for probing molecular recognition and catalysis by enzymes was
demonstrated inasmuch as discriminating peptide substrates could be identified
for even closely related proteases or tyrosine kinases. Furthermore, the
modification of whole phage libraries by tyrosine kinases led to the identification
of phosphopeptides specific for Src-homology-2 (SH2)- and phosphotyrosine-binding
(PTB) domains, which are both structural and functional modules facilitating
substrate recognition by protein kinases, phosphatases or adapter molecules
involved in signal transduction.
[Back to top] A Novel
Cleavage Protocol For Use With Rf-Encoded Split Pool Synthesis Technology:
Product Cleavage and Collection in Standard 96 Well Format. C. J. Andres,
R. T. Swann, K. Grant-Young, Stanley V. D'Andrea and Milind S. Deshpande.
A novel protocol which employs commercially available, standard tools
and hardware has been developed. This protocol allows for cleavage and
collection of IRORI Microkan products in 96 well plate format. Typically,
640 compounds can be cleaved in a 4 hour time period using approximately
3 square feet of space. This protocol has been used successfully for the
liberation of thousands of individual compounds, in single compound per
well format from the solid phase. Additionally, this protocol is the first
example of making IRORI Microkan technology directly compatible with standard
96 position deep well blocks.
[Back to top] A Solid
Phase Approach to Substituted Pyrimidines and Their Conversion into Condensed
Heterocycles for Potential Use in Combinatorial Chemistry. Sanjay K. Srivastava,
W. Haq and P.M.S. Chauhan.
A novel general synthesis of substituted pyrimidine 3 has been carried
out on solid support. The C-atoms carring the cyano, amino, carboxamido,
as well as anchoring site have exploited to generate libraries of compounds
6-8,10,13,15,17, 19, 21, 23, 25 and 27. A novel strategy to cleave the
resin to resin-site unsubstituted system has been developed and it provides
5,6-disubstituted pyrimidines 6-8. In addition, synthesis of 2,5,6-trisubstituted
pyrimidines of prototype 10 were carried out by nucleophilic displacement
of the anchor by various amines. Further investigations were directed toward
the solid phase synthesis of pyrimido[4,5-d]pyrimidines 12,16, 20 and 24
in which C-atoms carring the oxo, thio, amino, anchoring site as well as
NH could be introduced as center of diversity to generate libraries of
compounds for potential use. 4-Aminopyrimido[4,5-d]pyrimidines 13 and 17
were obtained from fusion of 3a with urea or thiourea followed by cleavage
of support while 3-phenylpyrimido[4,5-d]pyrimidines 21 and 27 were synthesized
from cyclisation of 4 with phenyl isocyanate or isothiocyanate followed
by release of resin. 7-substituted pyrimido[4,5-d]pyrimidines 15,19, 23
and 27 were obtained by oxidation of 12,16, 20.and 24 followed by aminolytic
cleavage of support.
[Back to top] High Throughput
Liquid Chromatography-Mass Spectrometry Assessment of the Metabolic Activity
of Commercially Available Hepatocytes from 96-Well Plates. Gory W. Caldwell,
John A. Mosucci and Enrique Chacon.
We have assessed the metabolic activity of freshly isolated and commercially
preserved rat, monkey, and human primary hepatocytes in a 96 well plate
format utilizing eight b-adrenolytic drugs as
model compounds. Sample introduction from 96 well plates, HPLC solvent
delivery, mass spectrometric (MS) detection, and/or UV detection were fully
integrated and operated unattended. After drugs were incubated with hepatocytes
for three or six hr, LC-MS analyses were carried out to determine the amount
of drug which was not metabolized. Two LC-MS methods were used which had
a sample throughput of 4 samples/hr and 12 samples/hr. Under optimal conditions,
this hepatic assay could screen 300 samples/week or 1200 samples/month.
Although freshly isolated hepatocytes were more active, commercially available
rat, monkey, and human primary isolated hepatocytes metabolized drug substrates
in similar relative rank orders. This drug-hepatocyte assay provides useful
information for prioritizing pharmaceutical leads in relative rank orders
or in a high/low manner according to their resistance toward liver metabolism.