Combinatorial Chemistry & High
Throughput Screening, Vol. 8, No. 4, 2005
Contents
Screening for Modulators of G Protein-Coupled Receptors
Guest Editor: Guido J.R.
Zaman
Editorial
Pp.283-283
Guido J.R. Zaman
[Abstract]
G-Protein
Coupled Receptor Assays: To Measure Affinity or Efficacy that is the Question Pp.285-292 C.
Williams and A. Sewing The
CellCard™ System: A Novel Approach to Assessing Compound Selectivity for Lead Prioritization
of G Protein-Coupled Receptors Pp.293-299 Oren
Beske, Simon Goldbard and Pierre Turpin High
Content Screening for G Protein-Coupled Receptors Using Cell-Based Protein
Translocation Assays Pp.301-309 Charlotta
Granas, Betina Kerstin Lundholt, Arne Heydorn, Viggo Linde, Hans-Christian
Pedersen, Christian Krog-Jensen, Mette M. Rosenkilde and Len Pagliaro Functional
G Protein-Coupled Receptor Assays for Primary and Secondary Screening Pp.311-318 Richard
M. Eglen General
Articles Using
IMAP Technology to Identify Kinase Inhibitors: Comparison with a Substrate
Depletion Approach and Analysis of the Nature of False Positives Pp.319-325 P.
Singh, B. Lillywhite, C. Bannaghan and P. Broad Substrate
Specificity and Novel Selective Inhibitors of TNF-a Converting Enzyme (TACE) from
Two-Dimensional Substrate Mapping Pp.327-339 Millard
H. Lambert, R. Kevin Blackburn, Theresa D. Seaton, Daniel B. Kassel, Daniel S.
Kinder, M. Anthony Leesnitzer, D. Mark Bickett, Janet R. Warner, Marc W.
Andersen, Jennifer G. Badiang, David J. Cowan, Michael D. Gaul, Kimberly G.
Petrov, Michael H. Rabinowitz, Robert W. Wiethe, J.
David Becherer, Darryl L. McDougald, David L. Musso, Robert C. Andrews and
Marcia L. Moss Rat
PXR Reporter-Gene Activity Correlates with the Induction of CYP3A in Rat
Precision-Cut Liver Slices Pp.341-346 Xiaoming
Cui, Ann Thomas, Diana Montgomery, Chunyan Gu, Richard A. Morrison, Ronald E.
White and K.-C. Cheng Identification
of Nitric Oxide Donors by Biomimetic HTS Application Pp.347-352 Gyorgy
T. Balogh, Balazs Dalmadi, Attila Bielik and Gyorgy M. Keseru High
Throughput Coagulation Analyzers Review Pp.353-360 M.H.
Qari Recent
Advances in Analytical Construct Resins Pp.361-371 D.
M. Whitehead, S.C. McKeown and A Routledge Meet
the Guest Editor Pp.373-373 Guido
J.R. Zaman Abstracts [Back to top] Editorial Guido J.R. Zaman G protein-coupled receptors
(GPCRs) represent the largest super family of all receptor types, and account
for the major proportion of current drug targets. Many pharmaceutical
industries have embarked on high-throughput screening to identify new low
molecular weight modulators for GPCRs. Many assay strategies for identification
of hits from screening are available. These include assays that address the
affinity of compounds for certain receptors, i.e. radioligand binding assays,
as well as assays that permit detection of ligands with a certain desired
functional effect, such as agonism, antagonism or inverse agonism. The choice
of the right assay format strongly impacts on the probability of success of a
screening campaign, and sets the direction of the subsequent hit optimization
program. Therefore, assays and screening strategies have to be smart. In this
mini-hot topic, four papers on assay development and screening strategies for
GPCRs have been collected. In: “G protein-coupled receptor assays:
To measure affinity or efficacy that is the question,” Christine Williams and
Andreas Sewing discuss the practical and theoretical considerations of
employing cell-based assays, using examples from the screening portfolio at
Pfizer, Sandwich (U.K.). Oren Beske and colleagues from Vitra Bioscience
describe a novel technology that allows multiple GPCRs to be assayed
simultaneously using the same concentration of compound from the exact same
compound source. Their technology may fulfill the need within pharmaceutical
industry to understand selectivity at an earlier stage of drug discovery. In addition to the classical coupling and signaling
through G proteins, GPCRs demonstrate various other types of behavior [1]. One
is receptor internalization. Charlotta Grånäs and colleagues from BioImage A/S
present a case-study on the internalization screening of the chemokine receptor
CXCR4 using image-based, high-content screening assay technology. Interesting
preliminary data show that compounds can be identified that induce receptor
internalization, but with little effect on CXCR4 signaling. Finally, a
comprehensive overview of functional cell-based assay approaches for GPCRs is
provided by Richard Eglen from DiscoveRx. Recent technological developments,
such as new assays to measure inositol phosphate second messengers, are
discussed in this review. I would like to thank the authors
for their contributions to this mini-hot topic. It has been a pleasure to work
with them. I hope you will enjoy reading their articles. [Back to top] C.
Williams and A. Sewing Cell-based assays have always played
an important role in the pharmaceutical industry, providing information about
the functional effects of compounds. These functional assays have traditionally
accompanied facile biochemical high throughput screening programmes, being
applied as secondary assays in the later stages of lead development. However,
with the disappointing reality that there is not likely to be a plethora of
novel, druggable targets in the post-genomic era, the role of cell-based assays
in drug discovery is beginning to change. Competition to develop the
"best" agents for well established targets and find more effective
ways of identifying "novel" agents is driving the industry towards a
"quality" versus "quantity" approach. Advances in genetic
engineering, automation compatible functional assay technologies and the
introduction of more sophisticated robotic systems, have facilitated the
application of cell-based assays to primary screening. However, despite some
apparent success to move these assays into the routine "toolbox" for
high throughput screening, certain preconceptions and concerns about cell-based
assays persist and the subject remains a topic of much debate. Here we use
examples from the screening portfolio at Pfizer, Sandwich, to discuss the
practical and theoretical considerations of employing cell-based assays in HTS
with a focus on G -protein coupled receptors. [Back to top] Advances in high throughput
screening technologies have led to the identification of many small molecules,
“hits”, with activities toward the target of interest. And, as the screening
technologies become faster and more robust, the rate at which the molecules are
identified continues to increase. This evolution of high throughput screening
technologies has generated a significant strain on the laboratories involved
with the downstream profiling of these hits using cell-based assays. The
CellCard™ System, by enabling multiple targets and/or cell lines to be assayed
simultaneously within a single well, provides a platform on which selectivity
screening can be quickly and robustly performed. Here we describe two case
studies using the b-lactamase and b-galactosidase reporter gene systems to
characterize G protein-coupled receptor agonist activity. Using these examples
we demonstrate how the implementation of this technology enables assay miniaturization
without micro-fluidic devices as well as how the inclusion of intra-well
controls can provide a means of data quality assessment within each well. [Back to top] Charlotta Granas, Betina Kerstin Lundholt,
Arne Heydorn, Viggo Linde, Hans-Christian Pedersen, Christian Krog-Jensen,
Mette M. Rosenkilde and Len Pagliaro G protein-coupled receptors
(GPCRs) have been one of the most productive classes of drug targets for
several decades, and new technologies for GPCR-based discovery promise to keep
this field active for years to come. While molecular screens for GPCR receptor
agonist- and antagonist-based drugs will continue to be valuable discovery
tools, the most exciting developments in the field involve cell-based assays
for GPCR function. Some cell-based discovery strategies, such as the use of b-arrestin as a surrogate marker for GPCR
function, have already been reduced to practice, and have been used as valuable
discovery tools for several years. The application of high content cell-based
screening to GPCR discovery has opened up additional possibilities, such as
direct tracking of GPCRs, G proteins and other signaling pathway components
using intracellular translocation assays. These assays provide the capability
to probe GPCR function at the cellular level with better resolution than has
previously been possible, and offer practical strategies for more definitive
selectivity evaluation and counter-screening in the early stages of drug
discovery. The potential of cell-based translocation assays for GPCR discovery
is described, and proof-of-concept data from a pilot screen with a CXCR4 assay
are presented. This chemokine receptor is a highly relevant drug target which
plays an important role in the pathogenesis of inflammatory disease and also
has been shown to be a co-receptor for entry of HIV into cells as well as to
play a role in metastasis of certain cancer cells. [Back to top]
Screening technologies for G
protein-coupled receptors comprise two major approaches; homogeneous assays,
conducted in microtiter plate formats, and protein redistribution assays that
require imaged-based analysis using automated confocal systems. Generally, the
former are used in primary screening campaigns for lead identification, while
the latter are used in secondary screens for lead optimization. Homogeneous
assays measure changes in G protein-coupled receptor second messengers such as
cAMP, Ins P3 or calcium. Protein redistribution assays assess
internalization of the receptor ligand complex or translocation of G-protein
coupled receptor-associated proteins, such as b-arrestin.
In the present review functional cell based approaches are discussed,
emphasizing the variety of non radiometric technologies now in use for HTS. [Back to top] IMAP® is a
fluorescence polarisation-based assay method which can be applied to the
measurement of protein kinase activity. Using a model serine/threonine kinase
we found that IMAP generated a good assay window (Z’ > 0.8), was very
tolerant of DMSO, and was flexible with respect to sample processing (stopped
reactions were stable over a period of several days). Using a set of six low
molecular weight inhibitors of the kinase, we found a good correlation between
IMAP and scintillation proximity assay (SPA) potency data. IMAP, which measures
product accumulation, was compared in an HTS setting with a substrate depletion
method (luminescence-based measurement of ATP concentration). There was a
reasonable (~50%) overlap in primary hits from a 17,000 compound set, but more
apparent false positives were generated from the IMAP method. We followed up
the compounds that showed activity in the IMAP method but not in the
luminescence assay. Approximately 10% of these compounds displayed intrinsic
fluorescence, suggesting that they were false actives by virtue of intrinsic
spectroscopic properties. Compound activity by competition of phosphopeptide
binding to IMAP beads can occur with high concentrations of chelating
compounds, but did not occur with any of the false actives, suggesting that this
form of interference is rare. [Back to top] Millard H. Lambert, R. Kevin Blackburn,
Theresa D. Seaton, Daniel B. Kassel, Daniel S. Kinder, M. Anthony Leesnitzer,
D. Mark Bickett, Janet R. Warner, Marc W. Andersen, Jennifer G. Badiang, David
J. Cowan, Michael D. Gaul, Kimberly G. Petrov, Michael H. Rabinowitz, Robert W.
Wiethe, J. David Becherer, Darryl L. McDougald, David L. Musso, Robert C.
Andrews and Marcia L. Moss We report a systematic analysis
of the P1’ and P2’ substrate specificity of TNF-a
converting enzyme (TACE) using a peptide library and a novel analytical method,
and we use the substrate specificity information to design novel reverse
hydroxamate inhibitors. Initial truncation studies, using the amino acid
sequence around the cleavage site in precursor-TNF-a, showed that good turnover was obtained with the peptide
DNPLAQAVRSS- NH2. Based on this result, 1000 different peptide
substrates of the form Biotin-LAQA-P1’-P2’- SSK(DNP)-NH2 were
prepared, with 50 different natural and unnatural amino acids at P1’ in
combination with 20 different amino acids at P2’. The peptides were pooled,
treated with purified microsomal TACE, and the reaction mixtures were passed
over a streptavidin affinity column to remove unreacted substrate and the
Nterminal biotinylated product. C-terminal cleavage products not binding to
streptavidin were subjected to liquid chromatography/mass spectrometry analysis
where individual products were identified and semiquantitated. 25 of the
substrates were resynthesized as discrete peptides and assayed with recombinant
TACE. The experiments show that recombinant TACE prefers lipophilic amino acids
at the P1’ position, such as phenylglycine, homophenylalanine, leucine and
valine. At the P2’ position, TACE can accommodate basic amino acids, such as
arginine and lysine, as well as certain non-basic amino acids such as citrulline,
methionine sulfoxide and threonine. These substrate preferences were used in
the design of novel reverse hydroxamate TACE inhibitors with phenethyl and
5-methyl-thiophene-methyl side-chains at P1’, and threonine and nitro-arginine
at P2’. [Back to top] Xiaoming Cui, Ann Thomas, Diana Montgomery,
Chunyan Gu, Richard A. Morrison, Ronald E. White and K.-C. Cheng Recent studies have suggested
that both constitutive androstane receptor (CAR) and pregnane Xreceptor (PXR)
are involved in the induction of rat liver microsomal cytochrome P-450 (CYP) 2B
and 3A through a mechanism called cross-talk. In this study we intend to determine
if a PXR-reporter gene assay could be used for the prediction of CYP3A and/or
CYP2B induction in rats. The induction of rat CYP2B and CYP3A by nineteen
structurally diverse compounds was evaluated by using rat precision-cut liver
slices and a rat PXR reporter-gene system. Induction of CYP2B and CYP3A mRNAs
in rat liver slices was quantified by real-time polymerase chain reaction. Rat
PXR activation was measured by induction of luciferase activity in rat PXR
reporter-gene system. Linear regression analysis of the fold of induction of
mRNA in liver slices and the fold of luciferase activity in rat PXR
reporter-gene system shows that a reasonable correlation (r2=0.6)
exists between the CYP3A induction and the rat PXR activation. A much lower
correlation was observed between CYP2B induction and the rat PXR activation (r2=0.1).
The results from this study suggest that the PXR may play a major role in the
induction of rat CYP3A, but not CYP2B. Therefore, the PXR-reporter gene assay
may be useful in a high-throughput screening to predict CYP3A induction in
rats. [Back to top] Metalloporphyrin catalyzed
biomimetic oxidation was used for the identification of nitric oxide (NO)
donors with diverse chemical structure. Methodology was validated by testing
known NO donors. Efficient automation of the test allowed us to investigate a subset
of our corporate library. Several hits identified in this campaign were
validated in both the chemical and also microsomal model that revealed all hits
to be active in the biological system, as well. One of the hits showed
comparable activity to V-PYRRO/NO, the prototypic liver selective NO donor. [Back to top]
The increasing no. of ordered
coagulation screening tests has led to a remarkable improvement in the efficiency
of automated coagulation testing, and appearance of a new generation of high
throughput analyzers ensuring accuracy and precision, and reducing the strain
and human error. The earliest analyzers operated
mechanically, using the hook to detect the clot in the cuvette are now replaced
with more sophisticated analyzer that simultaneously uses the clotting,
chromogenic, and immunological principles, to provide a versatile test menu
covering antigenic and functional aspects of coagulation. The armamentarium of coagulation
testing is complimented with tools like; automated platelet function analyzer,
flowcytometer, and molecular techniques including the polymerase chain
reaction, and lately the microarrary (biochip) technology. This review
addresses the principle of operation and distinctive features of various
automated high throughput coagulation analyzers, their impact on improving
efficiency, eliminating preanalytical and postanalytical handling thus
maximizing productivity and return on investment. [Back to top]
The area of solid-phase synthesis
has witnessed exponential growth in the last fifteen years, but difficulties associated
with the monitoring and analysis of resin-bound reactions and products have
been apparent due to a limited number of analytical methods available. With the
substrate tethered to an insoluble support traditional chromatographic
monitoring is only possible after cleavage. In order to address this
‘analytical bottleneck’ Geysen, in 1996 elaborated Merrifield’s initial dual
linker strategy by incorporating an encoding system between two in-line
linkers. These analytical construct resins represented a new approach for both
the quality control of solid-phase combinatorial libraries and for the
development of new synthetic sequences on solid-support. This review will
summarize the development and application of analytical construct resins
focusing on recent applications of the technology. [Back to top]
Dr. Guido Zaman is Section Head
Molecular in the Pharmacology Unit of N.V. Organon in Oss, the Netherlands. His
section is responsible for assay development from cDNA to HTS, and in vitro
biological testing from hit to clinical candidate. Guido completed his PhD in
molecular biology on protein – RNA interaction at the University of Nijmegen in
1991, and studied multidrug resistance of human cancer cells at the Netherlands
Cancer Institute in Amsterdam before joining Organon in 1996. In addition to
serving as a guest editor of the mini-hot topic in this issue, Guido Zaman is a
member of the Editorial Board of Assay & Drug Development Technologies.
Oren Beske, Simon Goldbard and
Pierre Turpin
Richard M. Eglen
P. Singh, B. Lillywhite, C.
Bannaghan and P. Broad
Gyorgy T. Balogh, Balazs Dalmadi,
Attila Bielik and Gyorgy M. Keseru
M.H. Qari
D. M. Whitehead, S.C. McKeown and A
Routledge
Guido J.R. Zaman