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Can human embryonic stem cells contribute to the discovery of safer and more effective drugs?
Can human embryonic stem cells contribute to the discovery of safer and more effective drugs? Gabriela Gebrin Cezar Few scientific achievements have received such irresistible attention from scientists, clinicians, and the general public as the ability of human embryonic stem (hES) cells to differentiate into functional cell types for regenerative medicine. The most immediate benefit of neurons, cardiomyocytes, and insulinsecreting cells derived from hES cells, however, may reside in their application in drug discovery and toxicology. The availability of renewable human cells with functional similarities to their in vivo counterparts is the first landmark for a new generation of cell-based assays. The development of cellbased assays using human cells that are physiological targets of drug activity will increase the robustness of target validation and efficacy, high-throughput screening (HTS), structure– activity relationship (SAR), and should introduce safer drugs into clinical trials and the marketplace. The pluripotency of embryonic stem cells, that is, the capacity to generate multiple cell types, is a novel path for the discovery of ‘regenerative drugs’, the pursuit of small molecules that promote tissue repair (neurogenesis, cardiogenesis) or proliferation of resident stem cells in different organs, thus creating drugs that work by a novel mechanism. Addresses Department of Animal Sciences, University of Wisconsin-Madison, 1675 Observatory Drive, Madison, WI 53706, United States Corresponding author: Cezar, Gabriela Gebrin ([email protected])
Current Opinion in Chemical Biology 2007, 11:405–409 This review comes from a themed issue on Chemical Biology and Stem Cells Edited by David Schaeffer Available online 26th July 2007 1367-5931/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.cbpa.2007.05.033
Introduction The progressive loss of pharmaceutical candidates from discovery through preclinical and clinical development, known as compound attrition, is a severe and continuous challenge to the pharmaceutical industry [1,2]. Compound attrition can be viewed as a complex system, influenced largely by failures in our ability to predict the toxicity and efficacy of pharmaceutical compounds to humans [3]. Another complicating factor to compound attrition is the identification of lead compounds through HTS in cellular substrates that are not in vivo targets of pharmacological activity, namely immortalized cell lines www.sciencedirect.com
such as human embryonic kidney cells (HEK 293) or Chinese hamster ovarian cells (CHO). The core benefit of hES cell technology to drug discovery and toxicology resides precisely in the fact that this renewable source of human cell types produces cell-based assays to improve our ability to predict human efficacy, toxicity, and identify chemical targets in their specific site of action rather than in ‘artificial’ biological environments. As a result, we expect a significant and positive impact of hES cell technology on the soaring compound attrition rates and escalating costs of drug discovery and development [3]. This review will focus primarily on scientific achievements over the past three years that effectively demonstrate the potential or direct application of stem cells in well-established standard assays in drug discovery and toxicology; HTS and measurement of cardiac ion-channel activity are typical examples of well-established assays. In addition, as stem cell scientists have an innate vision for the future, the review also explores opportunities and strategic convergences between embryonic stem cell technology and innovative platforms in chemical biology, namely the discovery of chemical entities with regenerative medicine properties and the assessment of pharmaceuticals using high-throughput molecular signatures, such as signalomics. Surprisingly, establishment of stem cells as an innovative platform for drug discovery and development is a nascent field [4,5] compared to parallel studies on stem cells for regenerative medicine. Overall, only approximately 2% of published studies on embryonic stem cells address predictive safety or efficacy of pharmaceuticals. Nonetheless, pharmaceutical sciences also benefit from research outcomes of regenerative medicine studies such as the establishment of functional dopaminergic neurons [6], cardiomyocytes [7], and pancreatic insulin-secreting cells [8] from human embryonic stem cells; these differentiated cell types, which are also targets of disease, can be readily integrated into the drug discovery and toxicology portfolio as screening tools. A new generation of cell-based assays is strongly warranted
Preclinical efficacy and toxicity testing are conducted largely in animal models as a means to validate the mechanism of action and predict adverse effects of compounds in human subjects. For developmental toxicity, for example, the most prevalent models that contribute to Current Opinion in Chemical Biology 2007, 11:405–409
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FDA approval of investigational new drugs are whole animal studies in rabbits and rats [9]. In vivo studies rely on administration of compounds to pregnant animals at different stages of pregnancy and embryonic/fetal development (first week of gestation, organogenesis stage, and full gestation length). However, these and other in vivo animal models are limited by a lack of robustness between animal and human responses to chemical compounds. Interspecies variability is a major culprit to soaring compound attrition rates [1,2,3]. Species differences are often manifested in trends such as dose sensitivity and pharmacokinetic processing of compounds. At present, animal models are approximately 50% efficient in predicting the toxicity of pharmaceuticals to the human heart, liver, and during human development [10]. Thus, human-directed predictive in vitro models present an opportunity to reduce the costs of new drug development and enable safer drugs. In vitro models have been employed in the drug industry for over 20 years throughout drug discovery and preclinical development [2,5,11]. Most of the current in vitro assays rely upon primary cell cultures or immortalized cell lines. While primary cell cultures are costly, time consuming, variable, have limited lifespan in vitro, and require the use of experimental animals, immortalized cell lines differ significantly from their in vivo counterparts in their ability to accurately assess efficacy and toxicity [1,2,5]. Thus, there is an obvious and significant demand to develop cell-based assays that are more predictive of human physiology and clinical response. Embryonic stem cells as a means to predict the toxicity of pharmaceutical compounds
The limited ability of current in vivo and in vitro assays to predict toxicity of pharmaceuticals to humans is the largest contributor to compound attrition [1,2]. In a similar fashion, the majority of published studies on the use of embryonic stem cells in drug discovery are focused on the development of in vitro models for predictive toxicity versus efficacy models for target validation. The pioneer initiative to integrate embryonic stem cells into predictive toxicology of pharmaceuticals was launched by the European Center for Validation of Alternative Methods (ECVAM) in the late nineties. Its primary outcome, the embryonic stem cell test (EST), has shown very promising results, with a 78% statistically significant correlation to in vivo studies, and the test was able to differentiate strong teratogens from moderate/weak or non-embryotoxic compounds [12]. This model is limited partly because toxicological endpoints are defined only for compounds that disrupt differentiation of heart cells during early development. It also fails to account for interspecies developmental differences between mice and humans, and thus fails to fully address the need for human-specific model systems. Subsequent efforts to the EST have been proposed to specifically Current Opinion in Chemical Biology 2007, 11:405–409
examine human developmental toxicity and neurotoxicity using more comprehensive approaches. Microarray analysis of human embryonic stem cells following exposure to known developmental disruptors is one approach under validation by ECVAM [13], while the neurotoxicity of different insults such as ischemia and toxic compounds was tested in neurons derived from both mouse and hES cells [14,15]. Here, predictive human neurotoxicity was determined by differential proteomic analysis with the ultimate goal of identifying biomarkers that are then employed to examine the neurotoxicity of new chemical entities (NCE). Our laboratory has taken one step further using chemical biology to predict the toxicity of pharmaceutical compounds during human development. We have pioneered the application of metabolomics to hES cells to examine biochemical pathways of developmental toxicity and discover predictive biomarkers of toxicity in hES cells and derivatives (Cezar, personal communication [16]). The metabolome is the dynamic set of small molecules present in a cell or tissue. Cardiotoxicity and hepatotoxicity remain the highest causes of drug safety liabilities and withdrawal of drugs during development and market launch [2]. Differentiation of mouse embryonic stem cells into cardiomyocytes followed by laser capture microdissection of beating foci and treatment with known agonists and antagonists of cardiac L-type calcium channels led to the development of an in vitro screen for cardiotoxicity [17]. Embryonic stem cell derived cardiomyocytes emulated in vivo response to cardiac L-type channel modulators such as nifedipine. The functional activity of multiple ion channels has also been verified in cardiomyocytes derived from human embryonic stem cells [7]; thus, these serve as a human substrate to screen for cardiotoxicity. The principal advantage of mouse and hES-derived cardiomyocytes is their ‘native’ environment with many ion channels for toxicity screening. Standard assays generally evaluate the effects of pharmaceuticals on single ion channels transfected into immortalized cells, such as the rapid inward rectifier potassium channel, known as hERG. The availability of a homogeneous source of human hepatocytes is considered a ‘holy grail’ for toxicity screening. In addition to hepatotoxicity, hES-derived hepatocytes would provide a renewable, cell-based assay to examine other key factors of compound attrition such as the metabolism of xenobiotics by CYP450 enzymes, drug–drug interactions, and the activity of drug transporters. Mouse hepatocyte-like cells were established from embryonic stem cells [18] with 70% of cells expressing the phenotypical marker albumin; hepatocyte-like cells also metabolized ammonia, lidocaine, and diazepam at approximately two-thirds the rate of primary mouse hepatocytes. The differences in liver physiology and drug metabolism between mice and humans are considerable, urging the development of hepatocytes from human www.sciencedirect.com
Can human embryonic stem cells contribute to the discovery of safer and more effective drugs? Cezar 407
stem cells. Recently, hepatocyte-like cells expressing alpha-fetoprotein, albumin, and hepatocyte nuclear factor 4alpha were differentiated from hES cells [19]. Additional functional phenotype analysis of these cells revealed induction of CYP450 enzymes, storage of glycogen, and transporter activity as demonstrated by influx and efflux of indocyanine green, a substrate of the transporter Pglycoprotein (PGP). A parallel study also reported glutathione S-transferase activity in hepatocyte-like cells derived from hES cells [20]. Altogether, these achievements are extremely encouraging findings in our quest to predict hepatotoxicity with greater confidence than that provided by current animal models. Establishment of in vitro models for target validation using human embryonic stem cells
Despite initial progress in embryonic stem cell based models for predictive toxicity, their applicability to increase confidence in mechanism, or efficacy, remain largely unexplored. Initial work in mouse and human embryonic stem cells highlighted the role of the canonical wingless (Wnt) pathway to maintain the stem cells undifferentiated, following inhibition of a widely explored pharmaceutical target, glycogen synthetase kinase 3beta (GSK3beta) [21]. Subsequent investigation using stem cells provided greater elucidation and target validation for GSK3beta antagonists. Both the exposure of mesenchymal stem cells to specific inhibitors of GSK3beta, which mimic Wnt signaling, and the modulation of this pathway in knockout mice enhanced osteogenesis in vitro, providing a potential target for bone formation [22,23]. More recently, a synergistic relationship between GSK3beta inhibition and activation of the type 5 metabotropic glutamate receptor (mGluR5) was demonstrated in mouse embryonic stem cells [24], a crucial finding for target validation in ongoing drug discovery programs. The establishment of functional dopaminergic neurons from human embryonic stem cells [6] was a major milestone to launch cell-based assays to validate the mechanism of action of pharmaceutical targets for Parkinson’s disease. A recent study was able to establish an in vitro model of Parkinson’s disease on the basis of differentiation and genetic modification of hES cells. Alphasynuclein is a well-established contributor to the ontogenesis Parkinson’s disease and a major component of Lewy bodies, the most typical pathology finding of this disorder [25]. Overexpression of alpha-synuclein and its mutations was achieved in terminally differentiating neuroectodermal cells derived from hES cells [26]. The findings in this in vitro model mirrored in vivo disease pathogenesis, as alpha-synuclein overexpression led to acute cytotoxicity and reduced the number of hESderived dopaminergic neurons. This is an important example of how hES cell technology contributes to in vitro models of disease for target validation. We also anticipate that another recent breakthrough of stem cell www.sciencedirect.com
research, namely the generation of large numbers of endocrine, pancreatic insulin-secreting cells from hES cells [9], will be invaluable to determine the efficacy of drug candidates for the treatment of diabetes. Strikingly, the insulin content of the hES-cell-derived insulinexpressing cells is similar to that of adult pancreatic islets [9]. This is the first renewable all human, non-immortalized, in vitro model for diabetes; thus, these cells may replace current cell-based models for diabetes drug discovery altogether in the future. Human embryonic stem cells and chemical biology: new frontiers in drug discovery
Pioneer studies screening high-content chemical libraries using embryonic stem cells were conducted by Ding et al. [27] with to identify small molecules controlling stem cell fate and elucidate signaling pathways associated with ‘stemness’. This approach has paved the way and seen initial accomplishments for a new era in HTS, where stem cells are the primary substrates for compound screening and their differentiated cells are the endpoints for lead identification [28,29]. Exposure of embryonic stem cells to small molecules such as cardiogenol-C, retinoic acid, and rosiglitazone induces directed differentiation of cardiomyocytes, neurons, and adipocytes, respectively [30,31]. Thus, HTS on human embryonic stem cells enables drug discovery for a new therapeutic area, regenerative medicine, which aims to repair damaged or diseased tissues and organs. Another small molecule, reversine, initially characterized for its ability to dedifferentiate myoblasts into multipotent progenitor cells [32], was recently shown to mediate de-differentiation of primary human fibroblasts into myogenic-competent cells both in vitro and in vivo [33]. It is also plausible to speculate that nascent HTS strategies will reveal small molecules capable of repairing damaged cells or tissue via recruitment and proliferation of stem cells within those tissues, such as neural stem cells found in the adult hippocampus [34] to ameliorate neurodegenerative disorders or cardiac stem cells [35] for ischemic heart disease. The ability of small molecules to induce the specific proliferation of tissue-specific stem cells has been demonstrated by small molecule inhibitors of GSK3beta, which augmented the population of hematopoetic stem cells in the bone marrow [36]. Signalomics is a high-throughput approach to elucidate simultaneous alterations in signal transduction cascades in response, for example, to a drug. A protein-fragment complementation assay that relies on interactions among proteins involved in a vast number of signaling pathways serves as the basis for this recently established cell-based assay [37]. The enticing aspect of this technology is that signal transduction in mammals is intrinsically composed of multiprotein complexes, which are uncommon targets or endpoints in current drug discovery strategies [38]. Ultimately, signalomics enables the identification of Current Opinion in Chemical Biology 2007, 11:405–409
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chemical–protein complexes that are responsible for both on-target as well as off-target drug effects. This will certainly enable identification of lead candidates with higher precision during lead-candidate optimization stages. The pioneer study by McDonald et al. [39] revealed novel mechanisms of actions for several of the 107 different tested drugs in addition to emulating known structure–function activities. We anticipate that replacement of HEK293 cells used in the study with human neurons, or other physiologically and clinically relevant cell type has the ability to greatly increase the power and selectivity of lead identification through this innovative chemical biology platform. Detection of hidden, unexpected, or off-target phenotypes during early stages of drug discovery due to chemical–protein interactions in the specific cell types of drug activity may significantly reduce compound attrition, enabling decision making on the progress or withdrawal of drug candidates in a much more timely and cost-effective manner.
Conclusion The integration of hES cell based assays into drug discovery and preclinical safety screening will dramatically benefit the pharmaceutical industry. Target validation, HTS, structure–activity relationship, identification and selection of lead compounds, and predictive toxicity studies can now be executed in renewable, scalable, nonimmortalized human cells that are the physiological target of compound activity with functional properties of their in vivo counterparts. So far the predominant focus of stem cell scientists has been on regenerative medicine applications and cell therapies; we anticipate a substantial shift of these research efforts toward development of cell-based assays for drug discovery in the nearest future. Humanized in vitro models will not only reduce compound attrition but importantly build reliability and robustness into the process of decision making with respect to the progress or withdrawal of drug candidates in the portfolio. Human embryonic stem cells will not replace, but perhaps refine the experimental use of animal models. In vivo studies are essential to examine the systemic effects of compound activity and toxicity that cannot be inferred using cell-based assays. Perhaps one of the most exciting research frontiers where human embryonic stem cells and chemical biology converge is the use of hES cells for screening of chemical libraries and the discovery of small molecules with the ability to regenerate or repair damaged cells and tissues. One of the pioneer reviews in the field was entitled ‘A role for chemistry in stem cell biology’ [31]. We propose that ‘A role for stem cell biology in chemistry’ is already in effect, as a result of two major aspects: firstly, the initial breakthroughs and unlimited opportunities for hES cells in predicting human response to chemicals and secondly the capacity of hES cells to serve as a new theme in drug discovery and change the way we think about ‘druggable targets’. Current Opinion in Chemical Biology 2007, 11:405–409
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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Preziosi P: Science, pharmacoeconomics and ethics in drug R&D: a sustainable future scenario? Nat Rev Drug Discov 2004, 3:521-526.
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Davila JC, Cezar GG, Thiede M, Strom S, Miki T, Trosko J: Use and application of stem cells in toxicology. Toxicol Sci 2004, 79:214-223.
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McNeish J: Embryonic stem cells in drug discovery. Nat Rev Drug Discov 2004, 3:70-80.
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Yan Y, Yang D, Zarnowska ED, Du Z, Werbel B, Valliere C, Pearce RA, Thomson JA, Zhang SC: Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 2005, 23:781-790.
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Kumar D, Kamp TJ, LeWinter MM: Embryonic stem cells: differentiation into cardiomyocytes and potential for heart repair and regeneration. Coron Artery Dis 2005, 16:111-116.
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D’Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick AD, Smart NG, Moorman MA, Kroon E, Carpenter MK, Baetge EE: Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol 2006, 24:1392-1401.
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Piersma AH: Alternative methods for developmental toxicity testing. Basic Clin Pharmacol Toxicol 2006, 98:427-431.
10. Greaves P, Williams A, Eve M: First dose of potential new medicines to humans: how animals help. Nat Rev Drug Discov 2004, 3:226-236. 11. Huuskonen H: New models and molecular markers in evaluation of developmental toxicity. Toxicol Appl Pharmacol 2005, 207(2 Suppl.):495-500. 12. Spielmann H, Seiler A, Bremer S, Hareng L, Hartung T, Ahr H, Faustman E, Haas U, Moffat GJ, Nau H et al.: The practical application of three validated in vitro embryotoxicity tests. The report and recommendations of an ECVAM/ZEBET workshop (ECVAM workshop 57). Altern Lab Anim 2006, 34:527-538. 13. Pellizzer C, Bremer S, Hartung T: Developmental toxicity testing from animal towards embryonic stem cells. ALTEX 2005, 22:47-57. 14. Schrattenholz A, Klemm M: Neuronal cell culture from human embryonic stem cells as in vitro model for neuroprotection. ALTEX 2007, 24:9-15. 15. Schrattenholz A, Wozny W, Klemm M, Schroer K, Stegmann W, Cahill MA: Differential and quantitative molecular analysis of ischemia complexity reduction by isotopic labeling of proteins using a neural embryonic stem cell model. J Neurol Sci 2005, 15:261-267. 16. Dutton G: Entering the Promising Field of Metabolomics. Genet Eng News 2007. 17. Chaudhary KW, Barrezueta NX, Bauchmann MB, Milici AJ, Beckius G, Stedman DB, Hambor JE, Blake WL, McNeish JD, Bahinski A, Cezar GG: Embryonic stem cells in predictive cardiotoxicity: laser capture microscopy enables assay development. Toxicol Sci 2006, 90:149-158. 18. Soto-Gutierrez A, Navarro-Alvarez N, Zhao D, Rivas-Carrillo JD, Lebkowski J, Tanaka N, Fox IJ, Kobayashi N: Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines. Nat Protoc 2007, 2(2):347-356. www.sciencedirect.com
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19. Hay DC, Zhao D, Ross A, Mandalam R, Lebkowski J, Cui W: Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities. Cloning Stem Cells 2007, 9:51-62. 20. Soderdahl T, Kuppers-Munther B, Heins N, Edsbagge J, Bjorquist P, Cotgreave I, Jernstrom B: Glutathione transferases in hepatocyte-like cells derived from human embryonic stem cells. Toxicol In Vitro 2007, 21:929-937 [Epub ahead of print]. 21. Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH: Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004, 10:55-63. 22. Gregory CA, Gunn WG, Reyes E, Smolarz AJ, Munoz J, Spees JL, Prockop DJ: How Wnt signaling affects bone repair by mesenchymal stem cells from the bone marrow. Ann NY Acad Sci 2005, 1049:97-106. 23. Tu X, Joeng KS, Nakayama KI, Nakayama K, Rajagopal J, Carroll TJ, McMahon AP, Long F: Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation. Dev Cell 2007, 12:113-127. 24. Spinsanti P, De Vita T, Di Castro S, Storto M, Formisano P, Nicoletti F, Melchiorri D: Endogenously activated mGlu5 metabotropic glutamate receptors sustain the increase in c-Myc expression induced by leukaemia inhibitory factor in cultured mouse embryonic stem cells. J Neurochem 2006, 99:299-307.
cells by a small molecule. Proc Natl Acad Sci USA 2006, 103:17266-17271. 29. Wu X, Walker J, Zhang J, Ding S, Schultz PG: Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway. Chem Biol 2004, 11:1229-1238. 30. Wu X, Ding S, Ding Q, Gray NS, Schultz PG: Small molecules that induce cardiomyogenesis in embryonic stem cells. J Am Chem Soc 2004, 126:1590-1591. 31. Ding S, Schultz PG: A role for chemistry in stem cell biology. Nat Biotechnol 2004, 22:833-840. 32. Chen S, Zhang Q, Wu X, Schultz PG, Ding S: Dedifferentiation of lineage-committed cells by a small molecule. J Am Chem Soc 2004, 126:410-411. 33. Anastasia L, Sampaolesi M, Papini N, Oleari D, Lamorte G, Tringali C, Monti E, Galli D, Tettamanti G, Cossu G, Venerando B: Reversine-treated fibroblasts acquire myogenic competence in vitro and in regenerating skeletal muscle. Cell Death Differ 2006, 13:2042-2051. 34. Warashina M, Min KH, Kuwabara T, Huynh A, Gage FH, Schultz PG, Ding S: A synthetic small molecule that induces neuronal differentiation of adult hippocampal neural progenitor cells. Angew Chem Int Ed Engl 2006, 45:591-593. 35. Urbanek K, Cesselli D, Rota M, Nascimbene A, De Angelis A, Hosoda T, Bearzi C, Boni A, Bolli R, Kajstura J et al.: Stem cell niches in the adult mouse heart. Proc Natl Acad Sci USA 2006, 103:9226-9231.
25. Gasser T: Genetics of Parkinson’s disease. Curr Opin Neurol 2005, 18:363-369.
36. Trowbridge JJ, Xenocostas A, Moon RT, Bhatia M: Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation. Nat Med 2006, 12:89-98.
26. Schneider BL, Seehus CR, Capowski EE, Aebischer P, Zhang SC, Svendsen CN: Over-expression of alpha-synuclein in human neural progenitors leads to specific changes in fate and differentiation. Hum Mol Genet 2007, 16:651-666.
37. Abraham RT: Signalomic signatures enlighten drug profiling. Nat Chem Biol 2006, 2:295-296.
27. Ding S, Wu TY, Brinker A, Peters EC, Hur W, Gray NS, Schultz PG: Synthetic small molecules that control stem cell fate. Proc Natl Acad Sci USA 2003, 100:7632-7637. 28. Chen S, Do JT, Zhang Q, Yao S, Yan F, Peters EC, Scholer HR, Schultz PG, Ding S: Self-renewal of embryonic stem
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38. MacDonald ML, Westwick JK: Exploiting network biology to improve drug discovery. Methods Mol Biol 2007, 356:221-232. 39. MacDonald ML, Lamerdin J, Owens S, Keon BH, Bilter GK, Shang Z, Huang Z, Yu H, Dias J, Minami T et al.: Identifying offtarget effects and hidden phenotypes of drugs in human cells. Nat Chem Biol 2006, 2:329-337.
Current Opinion in Chemical Biology 2007, 11:405–409
Current Opinion in Chemical Biology 2007, 11:405–409 This review comes from a themed issue on Chemical Biology and Stem Cells Edited by David Schaeffer Available online 26th July 2007 1367-5931/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.cbpa.2007.05.033
Introduction The progressive loss of pharmaceutical candidates from discovery through preclinical and clinical development, known as compound attrition, is a severe and continuous challenge to the pharmaceutical industry [1,2]. Compound attrition can be viewed as a complex system, influenced largely by failures in our ability to predict the toxicity and efficacy of pharmaceutical compounds to humans [3]. Another complicating factor to compound attrition is the identification of lead compounds through HTS in cellular substrates that are not in vivo targets of pharmacological activity, namely immortalized cell lines www.sciencedirect.com
such as human embryonic kidney cells (HEK 293) or Chinese hamster ovarian cells (CHO). The core benefit of hES cell technology to drug discovery and toxicology resides precisely in the fact that this renewable source of human cell types produces cell-based assays to improve our ability to predict human efficacy, toxicity, and identify chemical targets in their specific site of action rather than in ‘artificial’ biological environments. As a result, we expect a significant and positive impact of hES cell technology on the soaring compound attrition rates and escalating costs of drug discovery and development [3]. This review will focus primarily on scientific achievements over the past three years that effectively demonstrate the potential or direct application of stem cells in well-established standard assays in drug discovery and toxicology; HTS and measurement of cardiac ion-channel activity are typical examples of well-established assays. In addition, as stem cell scientists have an innate vision for the future, the review also explores opportunities and strategic convergences between embryonic stem cell technology and innovative platforms in chemical biology, namely the discovery of chemical entities with regenerative medicine properties and the assessment of pharmaceuticals using high-throughput molecular signatures, such as signalomics. Surprisingly, establishment of stem cells as an innovative platform for drug discovery and development is a nascent field [4,5] compared to parallel studies on stem cells for regenerative medicine. Overall, only approximately 2% of published studies on embryonic stem cells address predictive safety or efficacy of pharmaceuticals. Nonetheless, pharmaceutical sciences also benefit from research outcomes of regenerative medicine studies such as the establishment of functional dopaminergic neurons [6], cardiomyocytes [7], and pancreatic insulin-secreting cells [8] from human embryonic stem cells; these differentiated cell types, which are also targets of disease, can be readily integrated into the drug discovery and toxicology portfolio as screening tools. A new generation of cell-based assays is strongly warranted
Preclinical efficacy and toxicity testing are conducted largely in animal models as a means to validate the mechanism of action and predict adverse effects of compounds in human subjects. For developmental toxicity, for example, the most prevalent models that contribute to Current Opinion in Chemical Biology 2007, 11:405–409
406 Chemical Biology and Stem Cells
FDA approval of investigational new drugs are whole animal studies in rabbits and rats [9]. In vivo studies rely on administration of compounds to pregnant animals at different stages of pregnancy and embryonic/fetal development (first week of gestation, organogenesis stage, and full gestation length). However, these and other in vivo animal models are limited by a lack of robustness between animal and human responses to chemical compounds. Interspecies variability is a major culprit to soaring compound attrition rates [1,2,3]. Species differences are often manifested in trends such as dose sensitivity and pharmacokinetic processing of compounds. At present, animal models are approximately 50% efficient in predicting the toxicity of pharmaceuticals to the human heart, liver, and during human development [10]. Thus, human-directed predictive in vitro models present an opportunity to reduce the costs of new drug development and enable safer drugs. In vitro models have been employed in the drug industry for over 20 years throughout drug discovery and preclinical development [2,5,11]. Most of the current in vitro assays rely upon primary cell cultures or immortalized cell lines. While primary cell cultures are costly, time consuming, variable, have limited lifespan in vitro, and require the use of experimental animals, immortalized cell lines differ significantly from their in vivo counterparts in their ability to accurately assess efficacy and toxicity [1,2,5]. Thus, there is an obvious and significant demand to develop cell-based assays that are more predictive of human physiology and clinical response. Embryonic stem cells as a means to predict the toxicity of pharmaceutical compounds
The limited ability of current in vivo and in vitro assays to predict toxicity of pharmaceuticals to humans is the largest contributor to compound attrition [1,2]. In a similar fashion, the majority of published studies on the use of embryonic stem cells in drug discovery are focused on the development of in vitro models for predictive toxicity versus efficacy models for target validation. The pioneer initiative to integrate embryonic stem cells into predictive toxicology of pharmaceuticals was launched by the European Center for Validation of Alternative Methods (ECVAM) in the late nineties. Its primary outcome, the embryonic stem cell test (EST), has shown very promising results, with a 78% statistically significant correlation to in vivo studies, and the test was able to differentiate strong teratogens from moderate/weak or non-embryotoxic compounds [12]. This model is limited partly because toxicological endpoints are defined only for compounds that disrupt differentiation of heart cells during early development. It also fails to account for interspecies developmental differences between mice and humans, and thus fails to fully address the need for human-specific model systems. Subsequent efforts to the EST have been proposed to specifically Current Opinion in Chemical Biology 2007, 11:405–409
examine human developmental toxicity and neurotoxicity using more comprehensive approaches. Microarray analysis of human embryonic stem cells following exposure to known developmental disruptors is one approach under validation by ECVAM [13], while the neurotoxicity of different insults such as ischemia and toxic compounds was tested in neurons derived from both mouse and hES cells [14,15]. Here, predictive human neurotoxicity was determined by differential proteomic analysis with the ultimate goal of identifying biomarkers that are then employed to examine the neurotoxicity of new chemical entities (NCE). Our laboratory has taken one step further using chemical biology to predict the toxicity of pharmaceutical compounds during human development. We have pioneered the application of metabolomics to hES cells to examine biochemical pathways of developmental toxicity and discover predictive biomarkers of toxicity in hES cells and derivatives (Cezar, personal communication [16]). The metabolome is the dynamic set of small molecules present in a cell or tissue. Cardiotoxicity and hepatotoxicity remain the highest causes of drug safety liabilities and withdrawal of drugs during development and market launch [2]. Differentiation of mouse embryonic stem cells into cardiomyocytes followed by laser capture microdissection of beating foci and treatment with known agonists and antagonists of cardiac L-type calcium channels led to the development of an in vitro screen for cardiotoxicity [17]. Embryonic stem cell derived cardiomyocytes emulated in vivo response to cardiac L-type channel modulators such as nifedipine. The functional activity of multiple ion channels has also been verified in cardiomyocytes derived from human embryonic stem cells [7]; thus, these serve as a human substrate to screen for cardiotoxicity. The principal advantage of mouse and hES-derived cardiomyocytes is their ‘native’ environment with many ion channels for toxicity screening. Standard assays generally evaluate the effects of pharmaceuticals on single ion channels transfected into immortalized cells, such as the rapid inward rectifier potassium channel, known as hERG. The availability of a homogeneous source of human hepatocytes is considered a ‘holy grail’ for toxicity screening. In addition to hepatotoxicity, hES-derived hepatocytes would provide a renewable, cell-based assay to examine other key factors of compound attrition such as the metabolism of xenobiotics by CYP450 enzymes, drug–drug interactions, and the activity of drug transporters. Mouse hepatocyte-like cells were established from embryonic stem cells [18] with 70% of cells expressing the phenotypical marker albumin; hepatocyte-like cells also metabolized ammonia, lidocaine, and diazepam at approximately two-thirds the rate of primary mouse hepatocytes. The differences in liver physiology and drug metabolism between mice and humans are considerable, urging the development of hepatocytes from human www.sciencedirect.com
Can human embryonic stem cells contribute to the discovery of safer and more effective drugs? Cezar 407
stem cells. Recently, hepatocyte-like cells expressing alpha-fetoprotein, albumin, and hepatocyte nuclear factor 4alpha were differentiated from hES cells [19]. Additional functional phenotype analysis of these cells revealed induction of CYP450 enzymes, storage of glycogen, and transporter activity as demonstrated by influx and efflux of indocyanine green, a substrate of the transporter Pglycoprotein (PGP). A parallel study also reported glutathione S-transferase activity in hepatocyte-like cells derived from hES cells [20]. Altogether, these achievements are extremely encouraging findings in our quest to predict hepatotoxicity with greater confidence than that provided by current animal models. Establishment of in vitro models for target validation using human embryonic stem cells
Despite initial progress in embryonic stem cell based models for predictive toxicity, their applicability to increase confidence in mechanism, or efficacy, remain largely unexplored. Initial work in mouse and human embryonic stem cells highlighted the role of the canonical wingless (Wnt) pathway to maintain the stem cells undifferentiated, following inhibition of a widely explored pharmaceutical target, glycogen synthetase kinase 3beta (GSK3beta) [21]. Subsequent investigation using stem cells provided greater elucidation and target validation for GSK3beta antagonists. Both the exposure of mesenchymal stem cells to specific inhibitors of GSK3beta, which mimic Wnt signaling, and the modulation of this pathway in knockout mice enhanced osteogenesis in vitro, providing a potential target for bone formation [22,23]. More recently, a synergistic relationship between GSK3beta inhibition and activation of the type 5 metabotropic glutamate receptor (mGluR5) was demonstrated in mouse embryonic stem cells [24], a crucial finding for target validation in ongoing drug discovery programs. The establishment of functional dopaminergic neurons from human embryonic stem cells [6] was a major milestone to launch cell-based assays to validate the mechanism of action of pharmaceutical targets for Parkinson’s disease. A recent study was able to establish an in vitro model of Parkinson’s disease on the basis of differentiation and genetic modification of hES cells. Alphasynuclein is a well-established contributor to the ontogenesis Parkinson’s disease and a major component of Lewy bodies, the most typical pathology finding of this disorder [25]. Overexpression of alpha-synuclein and its mutations was achieved in terminally differentiating neuroectodermal cells derived from hES cells [26]. The findings in this in vitro model mirrored in vivo disease pathogenesis, as alpha-synuclein overexpression led to acute cytotoxicity and reduced the number of hESderived dopaminergic neurons. This is an important example of how hES cell technology contributes to in vitro models of disease for target validation. We also anticipate that another recent breakthrough of stem cell www.sciencedirect.com
research, namely the generation of large numbers of endocrine, pancreatic insulin-secreting cells from hES cells [9], will be invaluable to determine the efficacy of drug candidates for the treatment of diabetes. Strikingly, the insulin content of the hES-cell-derived insulinexpressing cells is similar to that of adult pancreatic islets [9]. This is the first renewable all human, non-immortalized, in vitro model for diabetes; thus, these cells may replace current cell-based models for diabetes drug discovery altogether in the future. Human embryonic stem cells and chemical biology: new frontiers in drug discovery
Pioneer studies screening high-content chemical libraries using embryonic stem cells were conducted by Ding et al. [27] with to identify small molecules controlling stem cell fate and elucidate signaling pathways associated with ‘stemness’. This approach has paved the way and seen initial accomplishments for a new era in HTS, where stem cells are the primary substrates for compound screening and their differentiated cells are the endpoints for lead identification [28,29]. Exposure of embryonic stem cells to small molecules such as cardiogenol-C, retinoic acid, and rosiglitazone induces directed differentiation of cardiomyocytes, neurons, and adipocytes, respectively [30,31]. Thus, HTS on human embryonic stem cells enables drug discovery for a new therapeutic area, regenerative medicine, which aims to repair damaged or diseased tissues and organs. Another small molecule, reversine, initially characterized for its ability to dedifferentiate myoblasts into multipotent progenitor cells [32], was recently shown to mediate de-differentiation of primary human fibroblasts into myogenic-competent cells both in vitro and in vivo [33]. It is also plausible to speculate that nascent HTS strategies will reveal small molecules capable of repairing damaged cells or tissue via recruitment and proliferation of stem cells within those tissues, such as neural stem cells found in the adult hippocampus [34] to ameliorate neurodegenerative disorders or cardiac stem cells [35] for ischemic heart disease. The ability of small molecules to induce the specific proliferation of tissue-specific stem cells has been demonstrated by small molecule inhibitors of GSK3beta, which augmented the population of hematopoetic stem cells in the bone marrow [36]. Signalomics is a high-throughput approach to elucidate simultaneous alterations in signal transduction cascades in response, for example, to a drug. A protein-fragment complementation assay that relies on interactions among proteins involved in a vast number of signaling pathways serves as the basis for this recently established cell-based assay [37]. The enticing aspect of this technology is that signal transduction in mammals is intrinsically composed of multiprotein complexes, which are uncommon targets or endpoints in current drug discovery strategies [38]. Ultimately, signalomics enables the identification of Current Opinion in Chemical Biology 2007, 11:405–409
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chemical–protein complexes that are responsible for both on-target as well as off-target drug effects. This will certainly enable identification of lead candidates with higher precision during lead-candidate optimization stages. The pioneer study by McDonald et al. [39] revealed novel mechanisms of actions for several of the 107 different tested drugs in addition to emulating known structure–function activities. We anticipate that replacement of HEK293 cells used in the study with human neurons, or other physiologically and clinically relevant cell type has the ability to greatly increase the power and selectivity of lead identification through this innovative chemical biology platform. Detection of hidden, unexpected, or off-target phenotypes during early stages of drug discovery due to chemical–protein interactions in the specific cell types of drug activity may significantly reduce compound attrition, enabling decision making on the progress or withdrawal of drug candidates in a much more timely and cost-effective manner.
Conclusion The integration of hES cell based assays into drug discovery and preclinical safety screening will dramatically benefit the pharmaceutical industry. Target validation, HTS, structure–activity relationship, identification and selection of lead compounds, and predictive toxicity studies can now be executed in renewable, scalable, nonimmortalized human cells that are the physiological target of compound activity with functional properties of their in vivo counterparts. So far the predominant focus of stem cell scientists has been on regenerative medicine applications and cell therapies; we anticipate a substantial shift of these research efforts toward development of cell-based assays for drug discovery in the nearest future. Humanized in vitro models will not only reduce compound attrition but importantly build reliability and robustness into the process of decision making with respect to the progress or withdrawal of drug candidates in the portfolio. Human embryonic stem cells will not replace, but perhaps refine the experimental use of animal models. In vivo studies are essential to examine the systemic effects of compound activity and toxicity that cannot be inferred using cell-based assays. Perhaps one of the most exciting research frontiers where human embryonic stem cells and chemical biology converge is the use of hES cells for screening of chemical libraries and the discovery of small molecules with the ability to regenerate or repair damaged cells and tissues. One of the pioneer reviews in the field was entitled ‘A role for chemistry in stem cell biology’ [31]. We propose that ‘A role for stem cell biology in chemistry’ is already in effect, as a result of two major aspects: firstly, the initial breakthroughs and unlimited opportunities for hES cells in predicting human response to chemicals and secondly the capacity of hES cells to serve as a new theme in drug discovery and change the way we think about ‘druggable targets’. Current Opinion in Chemical Biology 2007, 11:405–409
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