Combinatorial Chemistry & High Throughput Screening - Volume 8, Issue 4, 2005

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 Granas 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. REFERENCE [1] Kenakin, T. Annu. Rev. Pharmacol. Toxicol., 2002, 42, 349.

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.

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 β- lactamase and β-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.

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 β-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.

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 β-arrestin. In the present review functional cell based approaches are discussed, emphasizing the variety of non radiometric technologies now in use for HTS.

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.

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-α 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-α, 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'.

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.

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.

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.

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.

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.