- Email: [email protected]
Phospho-specific flow cytometry in drug discovery
Vol. 2, No. 3 2005
Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Emerging technologies
Phospho-specific flow cytometry in drug discovery Matthew R. Clutter, Peter O. Krutzik, Garry P. Nolan* Baxter Laboratory of Genetic Pharmacology, Department of Microbiology and Immunology, School of Medicine, Stanford University, 269 Campus Drive, CCSR 3205, Stanford, CA 94305, USA
Here we present phospho-specific flow cytometry as a new tool for drug discovery with applications throughout the drug development pipeline, from target identification
to
library
screening,
disease
model
Section Editors: Steve Gullans – RxGen, Inc., New Haven, CT, USA Robert Zivin – Johnson and Johnson, New Brunswick, NJ, USA
assessment and clinical screening and diagnostics. The single cell, multiparameter nature of flow cytometry generates high-content datasets, and current improvements in the technology are rapidly increasing its high-throughput capacity, making it a valuable platform in modern drug discovery.
Introduction It is clear that new ‘drug target’ identification nears an alltime high, and the speed with which initial drug ‘hits’ are created is radically increasing. But moving these drug leads through the clinic and to FDA approval still proceeds at the same snail’s pace. It has been suggested by many observers that higher success rates might come from the use of cellbased screens. Others have suggested that drug screens to achieve specific ‘pathway configurations’ might be more relevant and lead to higher specificity drugs with fewer offtarget toxicity complications. But can current drug screening approaches meet these needs while providing clinically relevant data? Are there approaches that merge drug-screening efforts with clear follow-up on drug effects in a clinical setting? In this review we discuss advantages of the recently developed PHOSPHO-SPECIFIC FLOW CYTOMETRY technique as a drug dis*Corresponding author: G.P. Nolan ([email protected]) 1740-6749/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2005.08.010
covery tool and its potential use in generating novel targets, performing cell-based screens, assessing disease model relevancy and facilitating patient monitoring and diagnostic stratification. In its most basic application, the technique enables the analysis of phosphorylation states in single fixed, permeabilized cells by staining samples with one or more fluorescently conjugated antibodies specific for phosphorylated protein epitopes. As an extension of SINGLE CELL analysis by flow cytometry, additional antibodies specific for cell surface identification markers can be utilized to resolve cell subtype-specific responses from complex cell populations [1]. Thus, like other cell-based assays, phospho-specific flow cytometry yields relevant data regarding a drug candidate’s permeability, but can do so with cells from a more clinical context. Unlike other cell-based screening techniques, phospho-specific flow cytometry produces MULTIPARAMETER data allowing one to effectively analyze cells in complex, heterogeneous populations such as primary human blood and murine immunological tissues [2–4]. In addition, data are provided at the single cell level, eliminating averaging effects inherent to traditional biochemical screening assays (Table 1). The approach provides a rapid method of profiling and characterizing human disease and a platform for analyzing how accurately cell lines and mouse models represent human disease at the signaling level. Following the details of such signaling through to clinical settings allows the same ‘high www.drugdiscoverytoday.com
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Glossary In vitro kinase assay: common high-throughput technique for assessing the ability of a purified kinase to phosphorylate a peptide or protein substrate in a cell-free environment. Multiparameter/multidimensional: the ability to measure multiple biochemical parameters in each cell by flow cytometry, enabling rapid multiplexing for dataset generation. Phospho-specific antibody: an antibody specific for a protein epitope only in the phosphorylated state. Monoclonal antibodies primarily conjugated to fluorescent dyes are optimal for use in phospho-specific flow cytometry. Phospho-specific flow cytometry: newly developed technique for assessing phosphorylation states of specific protein epitopes in fixed, permeabilized cells. Multiparameter, single cell characteristics distinguish the technique from many other cell-based kinase assays. Single cell: the ability to measure characteristics of individual cells by flow cytometry, permitting rapid high-content data collection and cell subset evaluation.
throughput assay’ to be applied to human clinical samples as was developed for the original screening. We anticipate that such mechanistic assessment of complex disease in highly relevant biological systems will advance drug discovery innovation through identification of novel targets. As we will discuss, phospho-specific flow cytometry can be applied at all stages of the drug development process, from target identification, cell-based screening and target validation in murine models or patient samples, through the phases of clinical trials, and even once a drug has reached the clinic to determine its efficacy in recipient patients. We propose that new technologies for drug screening must take advantage of single cell information to truly explore the range of variability across cell populations that exists in the patient.
Phosphorylation, kinases and a failure of the traditional drug discovery process? Reversible phosphorylation catalyzed by protein kinases mediate a significant portion of signal transduction in eukaryotic cells and as such are considered key regulators in cellular activities. Numerous diseases have been associated with dysregulated protein phosphorylation as demonstrated by one
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recent human genomics study in which 164 kinases were mapped to tumor-associated amplicons and 80 kinases were mapped to loci associated with other major diseases [5]. Additional cases of kinase overexpression or mutation in disease are found in autoimmunity, immunodeficiency, neural disorders and cardiovascular conditions. Consequently, protein kinases have become the second largest class of drug targets behind G-protein-coupled receptors [6]. Although kinases are attractive drug targets because of their integral role in many cellular processes, there are concerns regarding the feasibility of kinase-specific inhibition. These concerns arise in part because the mammalian genome contains many structurally similar protein kinases, and in part because nearly all kinase inhibitors target a highly conserved ATP-binding catalytic domain [7]. Clinical success of kinase inhibitors like imatinib (Gleevec), erlotinib (Tarceva) and gefitinib (Iressa) in treating various cancers, however, underscores that drugs against this class of targets can be effective [8]. Such success advocates a need for expanded and perhaps novel drug discovery efforts in identifying diseaserelevant biology of the protein kinase targets and their selective inhibitors. The current age of drug discovery has had high expectations, stemming primarily from promises that sequencing the human genome would provide a wealth of new and potentially ‘drug-able’ targets [9–11], as evidenced by the many kinases discovered by genomic sequencing. However, in spite of successes in human genomics, the pace of innovative drug discovery by the biomedical research community has disappointed many observers. In recent years, the number of original drug submissions to the US FDA and the European Medicines Evaluation Agency (EMEA) have declined and approval times have risen, all despite increased biomedical R&D spending and improved efficiency of the drug application review process [12–14]. Several explanations have been proposed regarding the supposed failure of current drug discovery efforts [12]. Some have suggested that the ‘easiest’ targets have already been discovered. Another explanation is that a sufficient amount of time and money have not yet been invested in drug
Table 1. Comparison of phosphorylation assay technologies Technology - Cell-free assays
- ‘Lysed’ cell-based assays
- ‘Whole’ cell-based assays
Example
- In vitro kinase
- Western blot - ELISA - Bead-based immunoassay
- Phospho-specific flow cytometry
Pros
- Ultra high throughput - High sensitivity - Good scalability
- High throughput - High sensitivity - Off-target effects detectable
-
Cons
- Off-target effects not detectable - Moderate scalability - Moderate throughput - Permeability not assessed - Moderate sensitivity - Cell subset specificity not assayed - Cell subset specificity not assayed - Moderate scalability
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Multiparameter/single cell (high-content, cell subset-specific analysis) Clinical applicability Off-target effects detectable Permeability assessed
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discovery since the sequencing of the human genome to meet the lofty expectations. Although these notions might be the actual contributors to decelerating drug discovery innovation, there is an additional explanation that biomedical researchers should carefully consider: in efforts to improve drug screening capabilities, complex human diseases are often simplified and attributed primarily to single protein aberrations, most notably cell surface receptors or intracellular signaling proteins like kinases. Three main biological assay systems – cell culture, traditional animal disease models and genetically manipulated mouse models – are surprisingly not subjected to sufficient crucial evaluation regarding their congruence with human biology. In one discussion of this, Kamb [15] speculates why failure rates of oncology drugs are so high in light of ‘faulty’ preclinical disease models. In another discussion, Horrobin [16] provides an interesting, and surely abbreviated, list of variables that could potentially differentiate in vivo biological behaviors of cells from corresponding in vitro phenomenology. To this end, our group recently observed differences between signal transduction in vitro and in vivo in the murine system [3]. This is not to say that current models of human biology are without merit because genetically manipulated mouse models have been highly informative in deciphering gene function [17]. However, it is becoming clear that, although important in making complex diseases tractable in the laboratory, over-simplification using disease models can hinder drug development progress, especially when laboratory models fail to reflect clinical realities. Contributing to this trend, the drug discovery industry, over the past several decades, has increasingly focused on cellfree, single target screening initiatives [18–20]. Indeed, the present era of high-throughput screening, beginning with an emergence of in vitro systems, marks a paradigm shift from a reliance on animal systems to a dependence on clinically proven targets, large chemical libraries, cell-free biochemical assays and robotic screens. The reduced complexity of these in vitro assays (e.g. IN VITRO KINASE ASSAYS) greatly increases the speed and sensitivity of compound screening and thereby facilitates identification of more lead drug candidates with increased chemical diversity. For instance, the most common kinase assays in drug discovery are high-throughput biochemical screens whereby large compound libraries are screened against known purified kinase targets [21]. In recent years significant progress has been made in advancing the speed and accuracy of more complex cell-based protein kinase assays [22,23], although these are mainly used in secondary screens where the importance of high-throughput is less emphasized. However, these cell-based models, often utilizing cell culture lines, might not represent the disease of interest accurately, and therefore they could yield hits that are incapable of treating the disease of interest in corresponding relevant primary cell subtypes.
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Because these high-throughput assay systems are based on prior knowledge of cellular function, they rarely generate drugs that act through novel biological mechanisms. This is illustrated by the fact that the target diversity of all marketed prescription products today is probably fewer than 100 molecular entities [17]. Underscoring this concept are emerging systems biology views, which hold that intercellular and intracellular signaling networks are complex, interdependent entities that can often compensate when forced into an unbalanced state. Therefore, the basic premise remains: How efficacious is it to perform in vitro screens on single proteins in the absence of the many regulators and upstream/ downstream components of a given pathway? As we will now discuss, phospho-specific flow cytometry has the potential to address: (1) the issue of diminished complexity inherent to cell-free assays, (2) important issues pertaining to the use of primary cells and even patient samples in drug discovery, and (3) the question of system relevance stemming from cellbased assays.
Phospho-specific flow cytometry Analysis of protein phosphorylation by flow cytometry has recently emerged as a powerful tool, particularly for the assessment of heterogeneous cell populations. Briefly, the technique is performed on cells in tissue culture, or on cells recently isolated from a patient, after activation of signal transduction (e.g. in vitro activation in the presence of an uncharacterized drug of interest). After an appropriate incubation period to allow signaling to occur, cells are fixed with paraformaldehyde (thus freezing the signaling system for subsequent analysis), permeabilized (to allow access for the staining antibodies) and then stained with fluorescently labeled antibodies raised against cell surface markers or specific intracellular epitopes of phosphorylated proteins (Fig. 1). The technique and methods of optimization have been described in detail elsewhere [2–4,24,25]. Two primary advantages of phospho-specific flow cytometry measurements as they pertain to drug discovery are the multiparameter nature of the data (Fig. 2A) and the single cell resolution (Fig. 2B). Multiparameter refers to an ability to simultaneously measure several biochemical parameters (e.g. via their antibody reactivity and fluorescence labeling) in each sample. For instance, staining panels consisting of fluorescently labeled antibodies against multiple cell surface markers and one or more intracellular phospho-protein epitopes are commonly used by our group for analyzing primary immune system cells [3,4]. With up to 13 dimensions assayable per cell [26], this generally permits analysis of all cell subtypes of interest and multiple intracellular phosphorylation state readouts. As an example, one could distinguish in a complex population of peripheral blood mononuclear cells the B cells, T cell, macrophages and monocytes, as well as multiple STAT or MAP kinase proteins that have been actiwww.drugdiscoverytoday.com
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Figure 1. Overview of the phospho-specific flow cytometry technique applied to drug discovery. Cultured or primary cells are aliquoted into 96-well plates and preincubated with a library of compounds. Cells are then stimulated in the presence of compound with various biological stimuli and fixed with formaldehyde to preserve signaling information. After permeabilization with methanol and staining with fluorescently labeled antibodies specific for cell surface markers or intracellular phosphorylated protein epitopes, flow cytometric analysis and electronic gating are used to assess phosphorylation status of proteins from cell subtypes of interest.
vated in these cell subsets – all at once. This represents a substantial advantage over traditional kinase assays with primary cells, which require time-consuming cell sorting or depletion steps before analysis to observe responses of homogeneous cell populations. As depicted in Fig. 2A, even after sorting or depletion, the analysis of multiple samples is required to yield the density of data derived from a single sample within a high dimensionality flow cytometry experiment. Therefore, in simultaneously assessing drug activities against several kinase targets within distinct cellular environments of multiple primary cell subtypes, multiparameter analysis lends phospho-specific flow cytometry a ‘highthroughput’ quality not accessed by other approaches. This is especially true if one believes that information regarding 298
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activities of fewer compounds on multiple targets (e.g. kinases) in multiple cell subtypes is at least as valuable as information regarding activities of larger numbers of compounds on single purified targets (given the limited identification of novel drug targets, this argument is certainly becoming more compelling). A second advantage of phospho-specific flow cytometry is the power of single cell measurements. This is particularly important when assessing heterogeneous cell populations because single cell data overcome averaging effects that are not resolved in traditional in vitro biochemical assays (Fig. 2B). In addition, this type of analysis is applicable to rare cell subsets because only several thousand cells of a particular subset are required to accurately assess phosphorylation state. The ability to analyze small cell numbers in accurately assessing kinase activity provided by this technique is therefore relevant when considering analysis of patient samples, which often contain limited cell numbers. Owing to its multiparameter, single cell nature, phosphospecific flow cytometry is uniquely suited to analyze cell subsets within complex populations of cells in parallel. This is perhaps the single most powerful aspect of the technology because it allows one to rapidly determine the efficacy of a drug in both the cell type of interest, and in several other cell types, simultaneously. Undoubtedly, many drugs fail in murine models or early clinical trials because of off-target effects, or particularly strong-targeted effects in non-diseased cells. Thus, opportunities are arising for new pharmacodynamic screens in a clinical setting, as well as new pharmacokinetic screens for drug concentration effects on cell targets in the patient. An additional application of phospho-specific flow cytometry, the elucidation of cell signaling networks, also depends upon the multiparameter, single cell nature of this technique [27]. As shown in this recent manuscript, the signaling phenotypes of primary human T cells – as assessed by flow cytometry and an advanced machine learning algorithm (termed Bayesian Networks) – allowed for reconstruction of complex signaling maps directly from raw flow cytometry data. The multiparameter correlated aspect of the data was important to driving the power of the algorithms in this latter approach. Thus, the data that are collected have high value in learning about real phenotypes of cells, which can greatly augment drug discovery and mechanistic understanding in primary and diseased patient cells.
Phospho-specific flow cytometry in the drug discovery pipeline Phospho-specific flow cytometry can be utilized in nearly every stage of drug discovery, from early screening to clinical trials and patient follow-up. Here we outline several stages of drug discovery and provide examples of how the technique can be used to provide data or information (Fig. 3, Panels I–IV
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Figure 2. Advantages of phospho-specific flow cytometry versus traditional biochemistry. (A) In analyzing complex cell populations like primary patient samples, the multidimensional nature of flow cytometry allows one to electronically gate different cell subtypes based on surface marker characteristics, and to analyze differential signaling behaviors of each of these subtypes from a single sample (bottom path). This is generally quicker than alternative biochemical methods (e.g. western blot) where one needs to mechanically sort pure populations of cells before analysis (top path). In addition, it is often difficult to sort pure populations of rare cell subtypes and recover enough material for analysis. (B) Traditional biochemical methods that measure averages of events are unable to resolve information from populations responding heterogeneously. Here Condition 1 and Condition 2 phosphorylation events are indistinguishable by western blot or in vitro kinase reaction analysis, although they appear distinct at the single cell level.
for Sections ‘Target identification’, ‘Cell-based chemical and target screening’, ‘Model assessment and utility’ and ‘Patient monitoring in clinical trials’, respectively).
Target identification Perhaps the most crucial aspect of drug discovery is finding the right target to modulate. Phospho-specific flow cytometry can be used in two different ways for target identification. First, the technique can be applied to analyze intracellular signaling cascade status of resting cells from a disease sample versus a normal sample. Although this is a classical approach, phospho-specific flow cytometry adds the aspects of single cell resolution and the ability to analyze small subsets of cells that might be at the root of a disease in the presence of large numbers of ‘bystander’ cells. A second application of the technique to target identification is by stimulating cells with various extracellular or cell permeable ligands, and to follow the response of the cell to these stimuli. In this way, disease samples are forced to respond and to ‘show their cards’ (i.e. to show if they are able to respond to a stimulus and whether the response is upregulated or abrogated). This latter application is yielding results in many cases where observation of basal signaling states proves inconclusive. Indeed, many diseases are driven
by irregular responses to extracellular stimuli that cause irregular growth, differentiation, or secretion. In this way, signal transduction pathways that are aberrantly responding to a crucial stimulus can be identified as drug ‘targets’ although they might not appear aberrantly regulated when resting in the absence of that stimulus.
Cell-based chemical and target screening Although flow cytometry might not be the most high throughput platform available for drug screening, it is extremely high content and can actually be used for screening large libraries. Plate loaders are now available for several flow cytometers that allow 96-well plates to be directly loaded and sampled for flow cytometry. Approximately 10,000 cells can be analyzed from each of 96 samples in 15 min. Therefore, with continuous running, about 10,000 samples can be analyzed in a 24-h period. Within a 2-week period, 100,000 compounds could easily be screened on a single ‘off the shelf’ cytometer. Other approaches are becoming available that greatly increase the throughput of these devices one or two orders of magnitude. However, the speed of sample acquisition is less important that the high content of data obtained per sample. Screens can be designed in which four to six phosphowww.drugdiscoverytoday.com
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Figure 3. Application of phospho-specific flow cytometry across the drug discovery pipeline. As shown in the top panel, the technique can be used to generate ‘phosphorylation profiles’ from stimulated normal and diseased patient samples, which can in turn be used to identify putative targets responsible for aberrant signaling. A hypothetical cell-based library screen is presented in the right-hand panel in which lead compounds are identified by flow cytometry (see histograms: green – stimulated control; red – stimulated in presence of lead compounds; gray – unstimulated) based on their ability to block phosphorylation of kinase 2 by kinase 1 and ‘re-route’ the network through kinase 3 as observed in normal patient signaling. The bottom panel exemplifies how the technique can be used to assess relevance of disease models. Here cells from the gray mouse demonstrate a similar profile to diseased patient samples and therefore this model is used for secondary in vivo screening of lead compounds. Finally, phospho-specific flow cytometry can be utilized, as suggested in the left-hand panel, in analysis of human blood samples from clinical trial patients to assess whether drug efficacy is maintained throughout treatment.
proteins are analyzed, perhaps in two to three cell types, in each well. Thus, although it might take 2 weeks to screen 100,000 compounds, data would be available concerning the effect of those compounds on four to six phosphoprotein targets in addition to cell-specific effects of each drug, effectively multiplying the data throughput 8–18fold. In addition, because these screens can be carried out in primary human cells, the chance of them providing relevant data greatly increases. It is becoming apparent to many researchers that high content is often favorable over high throughput in generating quality drug candidates. As a practical example of drug screening by phospho-specific flow cytometry, our group has recently generated data from primary cell screens revealing unexpected and fascinating differential responses to various compounds across cell subtypes (e.g. T cells, B cells, monocytes, among others) (POK and GPN, unpublished) that would have been missed had the assays been carried out on single cell lines or in a purely biochemical screen. 300
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Model assessment and utility Because phospho-specific flow cytometry can be used in target discovery, the identical assay used to find the target can be used to screen compounds. As mentioned previously, a crucial aspect of drug discovery is whether or not the assay represents the disease well, in function and phenotype. To this end, phospho-specific flow cytometry can be used to profile the signaling responses of several cell lines or murine disease models to determine how closely the model represents the overall signaling bio-signature of a known disease. It is crucial that the model not only represents the major defects responsible for disease but that it also shares other responsive or non-responsive patterns to stimuli because additional pathways might compensate for missing components or be synergistic in an unpredictable manner. In this way, a cell line or mouse model can be found that recapitulates the disease as accurately as possible without the inconvenience of using primary patient samples in high throughput or secondary cell-based screens.
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Patient monitoring in clinical trials Once a drug candidate reaches clinical trials, phospho-specific flow cytometry remains a powerful tool owing to its capability to analyze small numbers of cells or rare cell subsets in complex populations. For instance, in a clinical trial of a drug designed to inhibit ERK activity in B cells in response to abnormal levels of an extracellular ligand, one could acquire patient blood samples to (1) assess ERK phosphorylation levels in resting B cells, (2) stimulate with the extracellular ligand of interest to see whether the drug had been absorbed by the B cells, (3) determine the ERK phosphorylation levels of bystander cells (e.g. T cells, monocytes) under various conditions and (4) later treat with the drug of interest to determine whether cells have become non-responsive to the drug via mutation or receptor/target upregulation or downregulation. In addition to monitoring drug effects in patient blood samples, the technique will probably become a useful diagnostic tool in correlating signaling profiles from an individual patient to the efficacy of a particular drug in that patient. As an example, our group has applied phospho-specific flow cytometric profiling to stratify acute myelogenous leukemia (AML) patients according to their response to chemotherapy treatment [28]. This manuscript further demonstrated that multiple signaling subsets of cancer cells in AML existed and correlated with the most ‘discordant’ signaling phenotypes. Furthermore, those patients with many different signaling subsets of cells were those most probable to immediately fail chemotherapy because only some of these subsets of the cancer are killed by chemotherapy treatment. Thus, an opportunity arises for new assays in a clinical setting in the monitoring of novel drug effects on uniquely resistant cellular subsets (that represent novel disease targets in the patient). With more drugs being developed against specific intracellular targets, a technique such as phospho-specific flow cytometry is well suited to become an integral part of personalized medicine. In this way, patients can be treated with a drug, and its direct effects on the target of interest can be analyzed rapidly and efficiently, providing instant feedback on drug performance. This can help avoid costly and often devastating treatment regimens on patients that are nonresponsive at the single cell level.
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Links
http://www.proteomics.stanford.edu/nolan/ See BD PhosFlowTM pages: http://www.bd.com/ http://www.cellsignal.com/ http://www.phosphosite.org/ http://www.scbt.com/
particular cell subtypes in heterogeneous cell populations, such as immune system cells. Importantly, the single cell data provided are not subject to averaging effects of typical biochemical or population-based assays. Because diseases can be restricted to one or a few potentially rare cell subtypes, it is crucial to assess drug behavior in cells of interest as compared to their effects on bystander cells. As a cell-based assay, phospho-specific flow cytometry can generate drug candidates targeting unknown protein– protein interactions or undiscovered signaling pathway members that might act upstream or downstream of a target protein. At the very least these represent novel drug targets for future consideration via multiple drug discovery technologies like ultra-high-throughput single target screening. In addition, because of its potential to measure basal phosphorylation levels in disease and to generate broad signaling profiles of cell responsiveness to stimuli, the technique is ideally suited to assess the correlation and relevance of a cell line or mouse model to a human clinical condition. This will serve to increase the chances of finding more effective and relevant lead drug candidates. We believe that phosphospecific flow cytometry will become widely used in both basic R&D and clinical settings, thereby providing a consistent platform throughout the drug development process, from initial screens to personalized patient monitoring.
Outstanding issues Expanding the repertoire of PHOSPHO-SPECIFIC ANTIBODIES validated for use in phospho-specific flow cytometry. Further improving the throughput capacity of flow cytometry. Increasing the number of simultaneous phospho-specific parameters that can be robustly measured for a single cell. Standardizing/simplifying phospho-specific flow cytometry for a clinical setting.
Conclusion Although phospho-specific flow cytometry comprises a new class of technique for analysis of complex cellular phenotypes at the single cell level, it is clear it has applications at nearly all stages of drug discovery, from target identification through clinical trials. It will be possible to apply the technique to achieve a high-throughput quality with modern machinery and will probably yield high-content datasets from each sample. Because the platform facilitates multiparameter analysis, it allows for the interrogation of rare cell subsets or
References 1 Krutzik, P.O. et al. (2004) Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin. Immunol. 110, 206–221 2 Perez, O.D. and Nolan, G.P. (2002) Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat. Biotechnol. 20, 155–162 3 Krutzik, P.O. et al. (2005) Characterization of the murine immunological signaling network with phosphospecific flow cytometry. J. Immunol. 175, 2366–2373 www.drugdiscoverytoday.com
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4 Krutzik, P.O. et al. (2005) Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry. J. Immunol. 175, 2357–2365 5 Manning, G. et al. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934 6 Cohen, P. (2002) Protein kinases – the major drug targets of the twentyfirst century? Nat. Rev. Drug Discov. 1, 309–315 7 Bain, J. et al. (2003) The specificities of protein kinase inhibitors: an update. Biochem. J. 371 (Pt 1), 199–204 8 Green, M.R. (2004) Targeting targeted therapy. N. Engl. J. Med. 350, 2191– 2193 9 Broder, S. and Venter, J.C. (2000) Sequencing the entire genomes of freeliving organisms: the foundation of pharmacology in the new millennium. Annu. Rev. Pharmacol. Toxicol. 40, 97–132 10 Hopkins, A.L. and Groom, C.R. (2002) The druggable genome. Nat. Rev. Drug Discov. 1, 727–730 11 Drews, J. (1998) Biotechnology’s metamorphosis into a drug discovery industry. Nat. Biotechnol. 16 (Suppl.), 22–24 12 Drews, J. (2003) Strategic trends in the drug industry. Drug Discov. Today 8, 411–420 13 Reichert, J.M. (2003) Trends in development and approval times for new therapeutics in the United States. Nat. Rev. Drug Discov. 2, 695–702 14 DiMasi, J.A. et al. (2003) The price of innovation: new estimates of drug development costs. J. Health Econ. 22, 151–185 15 Kamb, A. (2005) What’s wrong with our cancer models? Nat. Rev. Drug Discov. 4, 161–165 16 Horrobin, D.F. (2003) Modern biomedical research: an internally selfconsistent universe with little contact with medical reality? Nat. Rev. Drug Discov. 2, 151–154
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18 19 20
21 22
23 24 25
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Zambrowicz, B.P. and Sands, A.T. (2003) Knockouts model the 100 best-selling drugs – will they model the next 100? Nat. Rev. Drug Discov. 2, 38–51 Kubinyi, H. (2003) Drug research: myths, hype and reality. Nat. Rev. Drug Discov. 2, 665–668 Butcher, E.C. (2005) Innovation: can cell systems biology rescue drug discovery? Nat. Rev. Drug Discov. 4, 461–467 Lombardino, J.G. and Lowe, J.A., III (2004) The role of the medicinal chemist in drug discovery – then and now. Nat. Rev. Drug Discov. 3, 853– 862 Walters, W.P. and Namchuk, M. (2003) Designing screens: how to make your hits a hit. Nat. Rev. Drug Discov. 2, 259–266 Russello, S.V. (2004) Assessing cellular protein phosphorylation: high throughput drug discovery technologies. Assay Drug Dev. Technol. 2, 225– 235 Giuliano, K.A. et al. (2003) Advances in high content screening for drug discovery. Assay Drug Dev. Technol. 1, 565–577 Perez, O.D. et al. (2004) Flow cytometric analysis of kinase signaling cascades. Methods Mol. Biol. 263, 67–94 Krutzik, P.O. and Nolan, G.P. (2003) Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 55, 61–70 De Rosa, S.C. and Roederer, M. (2001) Eleven-color flow cytometry. A powerful tool for elucidation of the complex immune system. Clin. Lab. Med. 21, 697–712, vii Sachs, K. et al. (2005) Causal protein-signaling networks derived from multiparameter single-cell data. Science 308, 523–529 Irish, J.M. et al. (2004) Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118, 217–228
Drug Discovery Today: Technologies Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Emerging technologies
Phospho-specific flow cytometry in drug discovery Matthew R. Clutter, Peter O. Krutzik, Garry P. Nolan* Baxter Laboratory of Genetic Pharmacology, Department of Microbiology and Immunology, School of Medicine, Stanford University, 269 Campus Drive, CCSR 3205, Stanford, CA 94305, USA
Here we present phospho-specific flow cytometry as a new tool for drug discovery with applications throughout the drug development pipeline, from target identification
to
library
screening,
disease
model
Section Editors: Steve Gullans – RxGen, Inc., New Haven, CT, USA Robert Zivin – Johnson and Johnson, New Brunswick, NJ, USA
assessment and clinical screening and diagnostics. The single cell, multiparameter nature of flow cytometry generates high-content datasets, and current improvements in the technology are rapidly increasing its high-throughput capacity, making it a valuable platform in modern drug discovery.
Introduction It is clear that new ‘drug target’ identification nears an alltime high, and the speed with which initial drug ‘hits’ are created is radically increasing. But moving these drug leads through the clinic and to FDA approval still proceeds at the same snail’s pace. It has been suggested by many observers that higher success rates might come from the use of cellbased screens. Others have suggested that drug screens to achieve specific ‘pathway configurations’ might be more relevant and lead to higher specificity drugs with fewer offtarget toxicity complications. But can current drug screening approaches meet these needs while providing clinically relevant data? Are there approaches that merge drug-screening efforts with clear follow-up on drug effects in a clinical setting? In this review we discuss advantages of the recently developed PHOSPHO-SPECIFIC FLOW CYTOMETRY technique as a drug dis*Corresponding author: G.P. Nolan ([email protected]) 1740-6749/$ ß 2005 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2005.08.010
covery tool and its potential use in generating novel targets, performing cell-based screens, assessing disease model relevancy and facilitating patient monitoring and diagnostic stratification. In its most basic application, the technique enables the analysis of phosphorylation states in single fixed, permeabilized cells by staining samples with one or more fluorescently conjugated antibodies specific for phosphorylated protein epitopes. As an extension of SINGLE CELL analysis by flow cytometry, additional antibodies specific for cell surface identification markers can be utilized to resolve cell subtype-specific responses from complex cell populations [1]. Thus, like other cell-based assays, phospho-specific flow cytometry yields relevant data regarding a drug candidate’s permeability, but can do so with cells from a more clinical context. Unlike other cell-based screening techniques, phospho-specific flow cytometry produces MULTIPARAMETER data allowing one to effectively analyze cells in complex, heterogeneous populations such as primary human blood and murine immunological tissues [2–4]. In addition, data are provided at the single cell level, eliminating averaging effects inherent to traditional biochemical screening assays (Table 1). The approach provides a rapid method of profiling and characterizing human disease and a platform for analyzing how accurately cell lines and mouse models represent human disease at the signaling level. Following the details of such signaling through to clinical settings allows the same ‘high www.drugdiscoverytoday.com
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Glossary In vitro kinase assay: common high-throughput technique for assessing the ability of a purified kinase to phosphorylate a peptide or protein substrate in a cell-free environment. Multiparameter/multidimensional: the ability to measure multiple biochemical parameters in each cell by flow cytometry, enabling rapid multiplexing for dataset generation. Phospho-specific antibody: an antibody specific for a protein epitope only in the phosphorylated state. Monoclonal antibodies primarily conjugated to fluorescent dyes are optimal for use in phospho-specific flow cytometry. Phospho-specific flow cytometry: newly developed technique for assessing phosphorylation states of specific protein epitopes in fixed, permeabilized cells. Multiparameter, single cell characteristics distinguish the technique from many other cell-based kinase assays. Single cell: the ability to measure characteristics of individual cells by flow cytometry, permitting rapid high-content data collection and cell subset evaluation.
throughput assay’ to be applied to human clinical samples as was developed for the original screening. We anticipate that such mechanistic assessment of complex disease in highly relevant biological systems will advance drug discovery innovation through identification of novel targets. As we will discuss, phospho-specific flow cytometry can be applied at all stages of the drug development process, from target identification, cell-based screening and target validation in murine models or patient samples, through the phases of clinical trials, and even once a drug has reached the clinic to determine its efficacy in recipient patients. We propose that new technologies for drug screening must take advantage of single cell information to truly explore the range of variability across cell populations that exists in the patient.
Phosphorylation, kinases and a failure of the traditional drug discovery process? Reversible phosphorylation catalyzed by protein kinases mediate a significant portion of signal transduction in eukaryotic cells and as such are considered key regulators in cellular activities. Numerous diseases have been associated with dysregulated protein phosphorylation as demonstrated by one
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recent human genomics study in which 164 kinases were mapped to tumor-associated amplicons and 80 kinases were mapped to loci associated with other major diseases [5]. Additional cases of kinase overexpression or mutation in disease are found in autoimmunity, immunodeficiency, neural disorders and cardiovascular conditions. Consequently, protein kinases have become the second largest class of drug targets behind G-protein-coupled receptors [6]. Although kinases are attractive drug targets because of their integral role in many cellular processes, there are concerns regarding the feasibility of kinase-specific inhibition. These concerns arise in part because the mammalian genome contains many structurally similar protein kinases, and in part because nearly all kinase inhibitors target a highly conserved ATP-binding catalytic domain [7]. Clinical success of kinase inhibitors like imatinib (Gleevec), erlotinib (Tarceva) and gefitinib (Iressa) in treating various cancers, however, underscores that drugs against this class of targets can be effective [8]. Such success advocates a need for expanded and perhaps novel drug discovery efforts in identifying diseaserelevant biology of the protein kinase targets and their selective inhibitors. The current age of drug discovery has had high expectations, stemming primarily from promises that sequencing the human genome would provide a wealth of new and potentially ‘drug-able’ targets [9–11], as evidenced by the many kinases discovered by genomic sequencing. However, in spite of successes in human genomics, the pace of innovative drug discovery by the biomedical research community has disappointed many observers. In recent years, the number of original drug submissions to the US FDA and the European Medicines Evaluation Agency (EMEA) have declined and approval times have risen, all despite increased biomedical R&D spending and improved efficiency of the drug application review process [12–14]. Several explanations have been proposed regarding the supposed failure of current drug discovery efforts [12]. Some have suggested that the ‘easiest’ targets have already been discovered. Another explanation is that a sufficient amount of time and money have not yet been invested in drug
Table 1. Comparison of phosphorylation assay technologies Technology - Cell-free assays
- ‘Lysed’ cell-based assays
- ‘Whole’ cell-based assays
Example
- In vitro kinase
- Western blot - ELISA - Bead-based immunoassay
- Phospho-specific flow cytometry
Pros
- Ultra high throughput - High sensitivity - Good scalability
- High throughput - High sensitivity - Off-target effects detectable
-
Cons
- Off-target effects not detectable - Moderate scalability - Moderate throughput - Permeability not assessed - Moderate sensitivity - Cell subset specificity not assayed - Cell subset specificity not assayed - Moderate scalability
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Multiparameter/single cell (high-content, cell subset-specific analysis) Clinical applicability Off-target effects detectable Permeability assessed
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discovery since the sequencing of the human genome to meet the lofty expectations. Although these notions might be the actual contributors to decelerating drug discovery innovation, there is an additional explanation that biomedical researchers should carefully consider: in efforts to improve drug screening capabilities, complex human diseases are often simplified and attributed primarily to single protein aberrations, most notably cell surface receptors or intracellular signaling proteins like kinases. Three main biological assay systems – cell culture, traditional animal disease models and genetically manipulated mouse models – are surprisingly not subjected to sufficient crucial evaluation regarding their congruence with human biology. In one discussion of this, Kamb [15] speculates why failure rates of oncology drugs are so high in light of ‘faulty’ preclinical disease models. In another discussion, Horrobin [16] provides an interesting, and surely abbreviated, list of variables that could potentially differentiate in vivo biological behaviors of cells from corresponding in vitro phenomenology. To this end, our group recently observed differences between signal transduction in vitro and in vivo in the murine system [3]. This is not to say that current models of human biology are without merit because genetically manipulated mouse models have been highly informative in deciphering gene function [17]. However, it is becoming clear that, although important in making complex diseases tractable in the laboratory, over-simplification using disease models can hinder drug development progress, especially when laboratory models fail to reflect clinical realities. Contributing to this trend, the drug discovery industry, over the past several decades, has increasingly focused on cellfree, single target screening initiatives [18–20]. Indeed, the present era of high-throughput screening, beginning with an emergence of in vitro systems, marks a paradigm shift from a reliance on animal systems to a dependence on clinically proven targets, large chemical libraries, cell-free biochemical assays and robotic screens. The reduced complexity of these in vitro assays (e.g. IN VITRO KINASE ASSAYS) greatly increases the speed and sensitivity of compound screening and thereby facilitates identification of more lead drug candidates with increased chemical diversity. For instance, the most common kinase assays in drug discovery are high-throughput biochemical screens whereby large compound libraries are screened against known purified kinase targets [21]. In recent years significant progress has been made in advancing the speed and accuracy of more complex cell-based protein kinase assays [22,23], although these are mainly used in secondary screens where the importance of high-throughput is less emphasized. However, these cell-based models, often utilizing cell culture lines, might not represent the disease of interest accurately, and therefore they could yield hits that are incapable of treating the disease of interest in corresponding relevant primary cell subtypes.
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Because these high-throughput assay systems are based on prior knowledge of cellular function, they rarely generate drugs that act through novel biological mechanisms. This is illustrated by the fact that the target diversity of all marketed prescription products today is probably fewer than 100 molecular entities [17]. Underscoring this concept are emerging systems biology views, which hold that intercellular and intracellular signaling networks are complex, interdependent entities that can often compensate when forced into an unbalanced state. Therefore, the basic premise remains: How efficacious is it to perform in vitro screens on single proteins in the absence of the many regulators and upstream/ downstream components of a given pathway? As we will now discuss, phospho-specific flow cytometry has the potential to address: (1) the issue of diminished complexity inherent to cell-free assays, (2) important issues pertaining to the use of primary cells and even patient samples in drug discovery, and (3) the question of system relevance stemming from cellbased assays.
Phospho-specific flow cytometry Analysis of protein phosphorylation by flow cytometry has recently emerged as a powerful tool, particularly for the assessment of heterogeneous cell populations. Briefly, the technique is performed on cells in tissue culture, or on cells recently isolated from a patient, after activation of signal transduction (e.g. in vitro activation in the presence of an uncharacterized drug of interest). After an appropriate incubation period to allow signaling to occur, cells are fixed with paraformaldehyde (thus freezing the signaling system for subsequent analysis), permeabilized (to allow access for the staining antibodies) and then stained with fluorescently labeled antibodies raised against cell surface markers or specific intracellular epitopes of phosphorylated proteins (Fig. 1). The technique and methods of optimization have been described in detail elsewhere [2–4,24,25]. Two primary advantages of phospho-specific flow cytometry measurements as they pertain to drug discovery are the multiparameter nature of the data (Fig. 2A) and the single cell resolution (Fig. 2B). Multiparameter refers to an ability to simultaneously measure several biochemical parameters (e.g. via their antibody reactivity and fluorescence labeling) in each sample. For instance, staining panels consisting of fluorescently labeled antibodies against multiple cell surface markers and one or more intracellular phospho-protein epitopes are commonly used by our group for analyzing primary immune system cells [3,4]. With up to 13 dimensions assayable per cell [26], this generally permits analysis of all cell subtypes of interest and multiple intracellular phosphorylation state readouts. As an example, one could distinguish in a complex population of peripheral blood mononuclear cells the B cells, T cell, macrophages and monocytes, as well as multiple STAT or MAP kinase proteins that have been actiwww.drugdiscoverytoday.com
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Figure 1. Overview of the phospho-specific flow cytometry technique applied to drug discovery. Cultured or primary cells are aliquoted into 96-well plates and preincubated with a library of compounds. Cells are then stimulated in the presence of compound with various biological stimuli and fixed with formaldehyde to preserve signaling information. After permeabilization with methanol and staining with fluorescently labeled antibodies specific for cell surface markers or intracellular phosphorylated protein epitopes, flow cytometric analysis and electronic gating are used to assess phosphorylation status of proteins from cell subtypes of interest.
vated in these cell subsets – all at once. This represents a substantial advantage over traditional kinase assays with primary cells, which require time-consuming cell sorting or depletion steps before analysis to observe responses of homogeneous cell populations. As depicted in Fig. 2A, even after sorting or depletion, the analysis of multiple samples is required to yield the density of data derived from a single sample within a high dimensionality flow cytometry experiment. Therefore, in simultaneously assessing drug activities against several kinase targets within distinct cellular environments of multiple primary cell subtypes, multiparameter analysis lends phospho-specific flow cytometry a ‘highthroughput’ quality not accessed by other approaches. This is especially true if one believes that information regarding 298
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activities of fewer compounds on multiple targets (e.g. kinases) in multiple cell subtypes is at least as valuable as information regarding activities of larger numbers of compounds on single purified targets (given the limited identification of novel drug targets, this argument is certainly becoming more compelling). A second advantage of phospho-specific flow cytometry is the power of single cell measurements. This is particularly important when assessing heterogeneous cell populations because single cell data overcome averaging effects that are not resolved in traditional in vitro biochemical assays (Fig. 2B). In addition, this type of analysis is applicable to rare cell subsets because only several thousand cells of a particular subset are required to accurately assess phosphorylation state. The ability to analyze small cell numbers in accurately assessing kinase activity provided by this technique is therefore relevant when considering analysis of patient samples, which often contain limited cell numbers. Owing to its multiparameter, single cell nature, phosphospecific flow cytometry is uniquely suited to analyze cell subsets within complex populations of cells in parallel. This is perhaps the single most powerful aspect of the technology because it allows one to rapidly determine the efficacy of a drug in both the cell type of interest, and in several other cell types, simultaneously. Undoubtedly, many drugs fail in murine models or early clinical trials because of off-target effects, or particularly strong-targeted effects in non-diseased cells. Thus, opportunities are arising for new pharmacodynamic screens in a clinical setting, as well as new pharmacokinetic screens for drug concentration effects on cell targets in the patient. An additional application of phospho-specific flow cytometry, the elucidation of cell signaling networks, also depends upon the multiparameter, single cell nature of this technique [27]. As shown in this recent manuscript, the signaling phenotypes of primary human T cells – as assessed by flow cytometry and an advanced machine learning algorithm (termed Bayesian Networks) – allowed for reconstruction of complex signaling maps directly from raw flow cytometry data. The multiparameter correlated aspect of the data was important to driving the power of the algorithms in this latter approach. Thus, the data that are collected have high value in learning about real phenotypes of cells, which can greatly augment drug discovery and mechanistic understanding in primary and diseased patient cells.
Phospho-specific flow cytometry in the drug discovery pipeline Phospho-specific flow cytometry can be utilized in nearly every stage of drug discovery, from early screening to clinical trials and patient follow-up. Here we outline several stages of drug discovery and provide examples of how the technique can be used to provide data or information (Fig. 3, Panels I–IV
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Figure 2. Advantages of phospho-specific flow cytometry versus traditional biochemistry. (A) In analyzing complex cell populations like primary patient samples, the multidimensional nature of flow cytometry allows one to electronically gate different cell subtypes based on surface marker characteristics, and to analyze differential signaling behaviors of each of these subtypes from a single sample (bottom path). This is generally quicker than alternative biochemical methods (e.g. western blot) where one needs to mechanically sort pure populations of cells before analysis (top path). In addition, it is often difficult to sort pure populations of rare cell subtypes and recover enough material for analysis. (B) Traditional biochemical methods that measure averages of events are unable to resolve information from populations responding heterogeneously. Here Condition 1 and Condition 2 phosphorylation events are indistinguishable by western blot or in vitro kinase reaction analysis, although they appear distinct at the single cell level.
for Sections ‘Target identification’, ‘Cell-based chemical and target screening’, ‘Model assessment and utility’ and ‘Patient monitoring in clinical trials’, respectively).
Target identification Perhaps the most crucial aspect of drug discovery is finding the right target to modulate. Phospho-specific flow cytometry can be used in two different ways for target identification. First, the technique can be applied to analyze intracellular signaling cascade status of resting cells from a disease sample versus a normal sample. Although this is a classical approach, phospho-specific flow cytometry adds the aspects of single cell resolution and the ability to analyze small subsets of cells that might be at the root of a disease in the presence of large numbers of ‘bystander’ cells. A second application of the technique to target identification is by stimulating cells with various extracellular or cell permeable ligands, and to follow the response of the cell to these stimuli. In this way, disease samples are forced to respond and to ‘show their cards’ (i.e. to show if they are able to respond to a stimulus and whether the response is upregulated or abrogated). This latter application is yielding results in many cases where observation of basal signaling states proves inconclusive. Indeed, many diseases are driven
by irregular responses to extracellular stimuli that cause irregular growth, differentiation, or secretion. In this way, signal transduction pathways that are aberrantly responding to a crucial stimulus can be identified as drug ‘targets’ although they might not appear aberrantly regulated when resting in the absence of that stimulus.
Cell-based chemical and target screening Although flow cytometry might not be the most high throughput platform available for drug screening, it is extremely high content and can actually be used for screening large libraries. Plate loaders are now available for several flow cytometers that allow 96-well plates to be directly loaded and sampled for flow cytometry. Approximately 10,000 cells can be analyzed from each of 96 samples in 15 min. Therefore, with continuous running, about 10,000 samples can be analyzed in a 24-h period. Within a 2-week period, 100,000 compounds could easily be screened on a single ‘off the shelf’ cytometer. Other approaches are becoming available that greatly increase the throughput of these devices one or two orders of magnitude. However, the speed of sample acquisition is less important that the high content of data obtained per sample. Screens can be designed in which four to six phosphowww.drugdiscoverytoday.com
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Figure 3. Application of phospho-specific flow cytometry across the drug discovery pipeline. As shown in the top panel, the technique can be used to generate ‘phosphorylation profiles’ from stimulated normal and diseased patient samples, which can in turn be used to identify putative targets responsible for aberrant signaling. A hypothetical cell-based library screen is presented in the right-hand panel in which lead compounds are identified by flow cytometry (see histograms: green – stimulated control; red – stimulated in presence of lead compounds; gray – unstimulated) based on their ability to block phosphorylation of kinase 2 by kinase 1 and ‘re-route’ the network through kinase 3 as observed in normal patient signaling. The bottom panel exemplifies how the technique can be used to assess relevance of disease models. Here cells from the gray mouse demonstrate a similar profile to diseased patient samples and therefore this model is used for secondary in vivo screening of lead compounds. Finally, phospho-specific flow cytometry can be utilized, as suggested in the left-hand panel, in analysis of human blood samples from clinical trial patients to assess whether drug efficacy is maintained throughout treatment.
proteins are analyzed, perhaps in two to three cell types, in each well. Thus, although it might take 2 weeks to screen 100,000 compounds, data would be available concerning the effect of those compounds on four to six phosphoprotein targets in addition to cell-specific effects of each drug, effectively multiplying the data throughput 8–18fold. In addition, because these screens can be carried out in primary human cells, the chance of them providing relevant data greatly increases. It is becoming apparent to many researchers that high content is often favorable over high throughput in generating quality drug candidates. As a practical example of drug screening by phospho-specific flow cytometry, our group has recently generated data from primary cell screens revealing unexpected and fascinating differential responses to various compounds across cell subtypes (e.g. T cells, B cells, monocytes, among others) (POK and GPN, unpublished) that would have been missed had the assays been carried out on single cell lines or in a purely biochemical screen. 300
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Model assessment and utility Because phospho-specific flow cytometry can be used in target discovery, the identical assay used to find the target can be used to screen compounds. As mentioned previously, a crucial aspect of drug discovery is whether or not the assay represents the disease well, in function and phenotype. To this end, phospho-specific flow cytometry can be used to profile the signaling responses of several cell lines or murine disease models to determine how closely the model represents the overall signaling bio-signature of a known disease. It is crucial that the model not only represents the major defects responsible for disease but that it also shares other responsive or non-responsive patterns to stimuli because additional pathways might compensate for missing components or be synergistic in an unpredictable manner. In this way, a cell line or mouse model can be found that recapitulates the disease as accurately as possible without the inconvenience of using primary patient samples in high throughput or secondary cell-based screens.
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Patient monitoring in clinical trials Once a drug candidate reaches clinical trials, phospho-specific flow cytometry remains a powerful tool owing to its capability to analyze small numbers of cells or rare cell subsets in complex populations. For instance, in a clinical trial of a drug designed to inhibit ERK activity in B cells in response to abnormal levels of an extracellular ligand, one could acquire patient blood samples to (1) assess ERK phosphorylation levels in resting B cells, (2) stimulate with the extracellular ligand of interest to see whether the drug had been absorbed by the B cells, (3) determine the ERK phosphorylation levels of bystander cells (e.g. T cells, monocytes) under various conditions and (4) later treat with the drug of interest to determine whether cells have become non-responsive to the drug via mutation or receptor/target upregulation or downregulation. In addition to monitoring drug effects in patient blood samples, the technique will probably become a useful diagnostic tool in correlating signaling profiles from an individual patient to the efficacy of a particular drug in that patient. As an example, our group has applied phospho-specific flow cytometric profiling to stratify acute myelogenous leukemia (AML) patients according to their response to chemotherapy treatment [28]. This manuscript further demonstrated that multiple signaling subsets of cancer cells in AML existed and correlated with the most ‘discordant’ signaling phenotypes. Furthermore, those patients with many different signaling subsets of cells were those most probable to immediately fail chemotherapy because only some of these subsets of the cancer are killed by chemotherapy treatment. Thus, an opportunity arises for new assays in a clinical setting in the monitoring of novel drug effects on uniquely resistant cellular subsets (that represent novel disease targets in the patient). With more drugs being developed against specific intracellular targets, a technique such as phospho-specific flow cytometry is well suited to become an integral part of personalized medicine. In this way, patients can be treated with a drug, and its direct effects on the target of interest can be analyzed rapidly and efficiently, providing instant feedback on drug performance. This can help avoid costly and often devastating treatment regimens on patients that are nonresponsive at the single cell level.
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Links
http://www.proteomics.stanford.edu/nolan/ See BD PhosFlowTM pages: http://www.bd.com/ http://www.cellsignal.com/ http://www.phosphosite.org/ http://www.scbt.com/
particular cell subtypes in heterogeneous cell populations, such as immune system cells. Importantly, the single cell data provided are not subject to averaging effects of typical biochemical or population-based assays. Because diseases can be restricted to one or a few potentially rare cell subtypes, it is crucial to assess drug behavior in cells of interest as compared to their effects on bystander cells. As a cell-based assay, phospho-specific flow cytometry can generate drug candidates targeting unknown protein– protein interactions or undiscovered signaling pathway members that might act upstream or downstream of a target protein. At the very least these represent novel drug targets for future consideration via multiple drug discovery technologies like ultra-high-throughput single target screening. In addition, because of its potential to measure basal phosphorylation levels in disease and to generate broad signaling profiles of cell responsiveness to stimuli, the technique is ideally suited to assess the correlation and relevance of a cell line or mouse model to a human clinical condition. This will serve to increase the chances of finding more effective and relevant lead drug candidates. We believe that phosphospecific flow cytometry will become widely used in both basic R&D and clinical settings, thereby providing a consistent platform throughout the drug development process, from initial screens to personalized patient monitoring.
Outstanding issues Expanding the repertoire of PHOSPHO-SPECIFIC ANTIBODIES validated for use in phospho-specific flow cytometry. Further improving the throughput capacity of flow cytometry. Increasing the number of simultaneous phospho-specific parameters that can be robustly measured for a single cell. Standardizing/simplifying phospho-specific flow cytometry for a clinical setting.
Conclusion Although phospho-specific flow cytometry comprises a new class of technique for analysis of complex cellular phenotypes at the single cell level, it is clear it has applications at nearly all stages of drug discovery, from target identification through clinical trials. It will be possible to apply the technique to achieve a high-throughput quality with modern machinery and will probably yield high-content datasets from each sample. Because the platform facilitates multiparameter analysis, it allows for the interrogation of rare cell subsets or
References 1 Krutzik, P.O. et al. (2004) Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin. Immunol. 110, 206–221 2 Perez, O.D. and Nolan, G.P. (2002) Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat. Biotechnol. 20, 155–162 3 Krutzik, P.O. et al. (2005) Characterization of the murine immunological signaling network with phosphospecific flow cytometry. J. Immunol. 175, 2366–2373 www.drugdiscoverytoday.com
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4 Krutzik, P.O. et al. (2005) Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry. J. Immunol. 175, 2357–2365 5 Manning, G. et al. (2002) The protein kinase complement of the human genome. Science 298, 1912–1934 6 Cohen, P. (2002) Protein kinases – the major drug targets of the twentyfirst century? Nat. Rev. Drug Discov. 1, 309–315 7 Bain, J. et al. (2003) The specificities of protein kinase inhibitors: an update. Biochem. J. 371 (Pt 1), 199–204 8 Green, M.R. (2004) Targeting targeted therapy. N. Engl. J. Med. 350, 2191– 2193 9 Broder, S. and Venter, J.C. (2000) Sequencing the entire genomes of freeliving organisms: the foundation of pharmacology in the new millennium. Annu. Rev. Pharmacol. Toxicol. 40, 97–132 10 Hopkins, A.L. and Groom, C.R. (2002) The druggable genome. Nat. Rev. Drug Discov. 1, 727–730 11 Drews, J. (1998) Biotechnology’s metamorphosis into a drug discovery industry. Nat. Biotechnol. 16 (Suppl.), 22–24 12 Drews, J. (2003) Strategic trends in the drug industry. Drug Discov. Today 8, 411–420 13 Reichert, J.M. (2003) Trends in development and approval times for new therapeutics in the United States. Nat. Rev. Drug Discov. 2, 695–702 14 DiMasi, J.A. et al. (2003) The price of innovation: new estimates of drug development costs. J. Health Econ. 22, 151–185 15 Kamb, A. (2005) What’s wrong with our cancer models? Nat. Rev. Drug Discov. 4, 161–165 16 Horrobin, D.F. (2003) Modern biomedical research: an internally selfconsistent universe with little contact with medical reality? Nat. Rev. Drug Discov. 2, 151–154
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18 19 20
21 22
23 24 25
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Zambrowicz, B.P. and Sands, A.T. (2003) Knockouts model the 100 best-selling drugs – will they model the next 100? Nat. Rev. Drug Discov. 2, 38–51 Kubinyi, H. (2003) Drug research: myths, hype and reality. Nat. Rev. Drug Discov. 2, 665–668 Butcher, E.C. (2005) Innovation: can cell systems biology rescue drug discovery? Nat. Rev. Drug Discov. 4, 461–467 Lombardino, J.G. and Lowe, J.A., III (2004) The role of the medicinal chemist in drug discovery – then and now. Nat. Rev. Drug Discov. 3, 853– 862 Walters, W.P. and Namchuk, M. (2003) Designing screens: how to make your hits a hit. Nat. Rev. Drug Discov. 2, 259–266 Russello, S.V. (2004) Assessing cellular protein phosphorylation: high throughput drug discovery technologies. Assay Drug Dev. Technol. 2, 225– 235 Giuliano, K.A. et al. (2003) Advances in high content screening for drug discovery. Assay Drug Dev. Technol. 1, 565–577 Perez, O.D. et al. (2004) Flow cytometric analysis of kinase signaling cascades. Methods Mol. Biol. 263, 67–94 Krutzik, P.O. and Nolan, G.P. (2003) Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 55, 61–70 De Rosa, S.C. and Roederer, M. (2001) Eleven-color flow cytometry. A powerful tool for elucidation of the complex immune system. Clin. Lab. Med. 21, 697–712, vii Sachs, K. et al. (2005) Causal protein-signaling networks derived from multiparameter single-cell data. Science 308, 523–529 Irish, J.M. et al. (2004) Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118, 217–228