Abstract
The last 10 years have seen advances in automation and high-throughput biochemistry in the drug-discovery arena. However, these advances have not led to improvements in drug-discovery success. Drug programs must find new ways to identify superior compounds. Advances in label-free assay technologies may provide advantages needed for improved drug discovery. In this article, we will discuss high-throughput MS, a technology that allows screening with native substrates and with targets inaccessible to standard assay formats. We will then discuss cell-based label-free biosensors, focusing on the increased information content available when using these platforms. We will conclude with speculation on the future and ways to obtain relevant biological information early in development to ensure the best compounds are promoted to medicinal chemistry campaigns.
Papers of special note have been highlighted as: ▪ of interest ▪▪ of considerable interest
Bibliography
- 1 Kenakin T, Miller LJ. Seven transmembrane receptors as shapeshifting proteins: the impact of allosteric modulation and functional selectivity on new drug discovery. Pharmacol. Rev.62(2),265–304 (2010).Crossref, Medline, CAS, Google Scholar
- 2 Smith RG, Sestili MA. Methods for ligand-receptor assays in clinical chemistry. Clin. Chem.26(5),543–550 (1980).Crossref, Medline, CAS, Google Scholar
- 3 Hart HE, Greenwald EB. Scintillation proximity assay (SPA)– a new method of immunoassay. Direct and inhibition mode detection with human albumin and rabbit antihuman albumin. Mol. Immunol.16(4),265–267 (1979).Crossref, Medline, CAS, Google Scholar
- 4 Udenfriend S, Gerber LD, Brink L, Spector S. Scintillation proximity radioimmunoassay utilizing 125I-labeled ligands. Proc. Natl Acad. Sci. USA82(24),8672–8676 (1985).Crossref, Medline, CAS, Google Scholar
- 5 Hoffman R, Cameron L. Characterization of a scintillation proximity assay to detect modulators of transforming growth factor α (TGFα) binding. Anal. Biochem.203,70–75 (1992).Crossref, Medline, CAS, Google Scholar
- 6 Jessop RA. Imaging proximity assays. Proc.SPIE3259,228–233 (1998).Crossref, CAS, Google Scholar
- 7 Watson J, Selkirk JV, Brown AM. Development of FlashplateTM technology to measure [35s]gtpys binding to chinese hamster ovary cell membranes expressing the cloned human 5-HTIB receptor. J. Biomol. Screen.3,101–105 (1998).Crossref, CAS, Google Scholar
- 8 Carpenter JW, Laethem C, Hubbard FR et al. Configuring radioligand receptor binding assays for HTS using scintillation proximity assay technology. Methods Mol. Biol.190,31–49 (2002).Medline, CAS, Google Scholar
- 9 Glickman JF, Schmid A, Ferrand S. Scintillation proximity assays in high-throughput screening. Assay Drug Dev.Technol.6(3),433–455 (2008).Crossref, Medline, CAS, Google Scholar
- 10 Schroeder KS, Neagle BD. FLIPR: a new instrument for accurate, high throughput optical screening. J. Biomolec. Screen.1(2),75–80 (1996).Crossref, CAS, Google Scholar
- 11 Glaser V. An interview with Kirk S. Schroeder, President, Essen Instruments. Assay Drug Develop. Technol.1,3–8 (2002).Crossref, Medline, Google Scholar
- 12 Ullman EF, Kirakossian H, Singh S et al. Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc. Natl Acad. Sci. USA91(12),5426–5430 (1994).Crossref, Medline, CAS, Google Scholar
- 13 Glickman JF, Wu X, Mercuri R et al. A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors. J. Biomol. Screen.7(1),3–10 (2002).Crossref, Medline, CAS, Google Scholar
- 14 Golla R, Seethala R. A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists. J. Biomol. Screen.7(6),515–525 (2002).Crossref, Medline, CAS, Google Scholar
- 15 Michael S, Auld D, Klumpp C et al. A robotic platform for quantitative high-throughput screening. Assay Drug. Dev. Technol.6(5),637–657 (2008).Crossref, Medline, CAS, Google Scholar
- 16 Hook DJ. Team building and leadership in the successful implementation of automation for high-throughput screening. J. Auto. Chem.18(4),131–134 (1996).Crossref, Medline, CAS, Google Scholar
- 17 Munos B. Lessons from 60 years of pharmaceutical innovation. Nat. Rev. Drug Discov.8(12),959–968 (2009).Crossref, Medline, CAS, Google Scholar
- 18 Kola I. The state of innovation in drug development. Clin. Pharmacol. Ther.83(2),227–230 (2008).Crossref, Medline, CAS, Google Scholar
- 19 Paolini GV, Shapland RH, van Hoorn WP, Mason JS, Hopkins AL. Global mapping of pharmacological space. Nat. Biotechnol.24(7),805–815 (2006).Crossref, Medline, CAS, Google Scholar
- 20 Hollinger MA. Introduction to Pharmacology (2nd Edition) Taylor and Francis, NY, USA, 6 (2003).Google Scholar
- 21 Kirschner MH. All Medicines are Poison! Author House, IN, USA, 4 (2009).Google Scholar
- 22 European research group of Morgan Stanley. Laboratory Equipment Magazine 12 March (2010).Google Scholar
- 23 Huggett B. Still strapped for cash. Nat. Biotech.27(6),493 (2009).Crossref, Medline, CAS, Google Scholar
- 24 Howitz KT, Bitterman KJ, Cohen HY, et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature425(6954),191–196 (2003).Crossref, Medline, CAS, Google Scholar
- 25 Milne JC, Lambert PD, Schenk S et al. Small molecule activators of SIRT1 as therapeutics for treatment of Type 2 diabetes. Nature450,712–716 (2007).Crossref, Medline, CAS, Google Scholar
- 26 Borra MT, Smith BC, Denu JM. Mechanism of human SIRT1 activation by resveratrol. J. Biol. Chem.280(17),17187–17195 (2005).Crossref, Medline, CAS, Google Scholar
- 27 Kaeberlein M, McDonagh T, Heltweg B et al. Substrate-specific activation of sirtuins by resveratrol. J. Biol. Chem.280(17),17038–17045 (2005).Crossref, Medline, CAS, Google Scholar
- 28 Pacholec M, Bleasdale JE, Chrunyk B et al. SRT1720, SRT2183, SRT1460, and Resveratrol are not direct activators of SIRT1. J. Biol. Chem.285,8340–8351 (2010).Crossref, Medline, CAS, Google Scholar
- 29 Beher D, Wu J, Cumine S et al. Resveratrol is not a direct activator of SIRT1 enzyme activity. Chem. Biol. Drug Des.74(6),619–624 (2009).Crossref, Medline, CAS, Google Scholar
- 30 Yan SF, King FJ, He Y, Caldwell JS, Zhou Y. Learning from the data: mining of large high-throughput screening databases. J. Chem. Inf. Model46(6),2381–2395 (2006).Crossref, Medline, CAS, Google Scholar
- 31 Xie XQ, Chen JZ. Data mining a small-molecule drug screening representative subset from NIH PubChem. J. Chem. Inf. Model48(3),465–475 (2008).Crossref, Medline, CAS, Google Scholar
- 32 Kostenis E, Waelbroeck M, Milligan G. Techniques: promiscuous Ga proteins in basic research and drug discovery. Trends Pharmacol. Sci.26(11),595–602 (2005).Crossref, Medline, CAS, Google Scholar
- 33 Zehender H, Le Goff F, Lehmann N, Filipuzzi I, Mayr LM. SpeedScreen: the ‘missing link’ between genomics and lead discovery. J. Biomol. Screen.9,498–505 (2004).Crossref, Medline, CAS, Google Scholar
- 34 Brown N, Zehender H, Azzaoui K, Schuffenhauer A, Mayr LM, Jacoby E. A chemoinformatics analysis of hit lists obtained from high-throughput affinity-selection screening. J. Biomol. Screen.11(2),123–130 (2006).Crossref, Medline, Google Scholar
- 35 Annis DA, Nazef N, Chuang CC, Scott MP, Nash HM. A general technique to rank protein–ligand binding affinities and determine allosteric versus direct binding site competition in compound mixtures. J. Am. Chem. Soc.126(47),15495–15503 (2004).Crossref, Medline, CAS, Google Scholar
- 36 Coburn CA, Stachel SJ, Li YM et al. Identification of a small molecule nonpeptide active site β-secretase inhibitor that displays a nontraditional binding mode for aspartyl proteases. J. Med. Chem.47(25),6117–6119 (2004).Crossref, Medline, CAS, Google Scholar
- 37 Annis DA, Nazef N, Chuang CC, Scott MP, Nash HM. A general technique to rank protein-ligand binding affinities and determine allosteric versus direct binding site competition in compound mixtures. J. Am. Chem. Soc.126(47),15495–15503 (2004).Crossref, Medline, CAS, Google Scholar
- 38 Whitehurst CE, Nazef N, Annis DA et al. Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry. J. Biomol. Screen.11(2),194–207 (2006).Crossref, Medline, Google Scholar
- 39 Annis DA, Shipps GW Jr, Deng Y et al. Method for quantitative protein-ligand affinity measurements in compound mixtures. Anal. Chem.79(12),4538–4542 (2007).Crossref, Medline, CAS, Google Scholar
- 40 Zheng H, Loh HH, Law PY. Agonist-selective signaling of G protein-coupled receptor: mechanisms and implications. IUBMB Life62(2),112–119 (2010).Medline, CAS, Google Scholar
- 41 Groebe DR. Screening for positive allosteric modulators of biological targets. Drug Discov. Today11(13–14),632–639 (2006).Crossref, Medline, CAS, Google Scholar
- 42 Groebe DR. In search of negative allosteric modulators of biological targets. Drug Discov. Today14(1–2),41–49 (2009).Crossref, Medline, CAS, Google Scholar
- 43 Roddy TP, Horvath CR, Stout SJ et al. Mass spectrometric techniques for label-free high-throughput screening in drug discovery. Anal. Chem.79(21),8207–8213 (2007).Crossref, Medline, CAS, Google Scholar
- 44 Zhang JH, Roddy TP, Ho PI et al. Assay development and screening of human DGAT1 inhibitors with an LC/MS-based assay: application of mass spectrometry for large-scale primary screening. J. Biomol. Screen.15(6),695–702 (2010) (Epub).Crossref, Medline, CAS, Google Scholar
- 45 Quercia AK, LaMarr WA, Myung J, Ozbal CC, Landro JA, Lumb KJ. High-throughput screening by mass spectrometry: comparison with the scintillation proximity assay with a focused-file screen of AKT1/PKB a. J. Biomol. Screen.12(4),473–480 (2007).Crossref, Medline, CAS, Google Scholar
- 46 Ozbal CC, LaMarr WA, Linton JR et al. High-throughput screening via mass spectrometry: a case study using acetylcholinesterase. Assay Drug Dev. Technol.2(4),373–381 (2004).Crossref, Medline, Google Scholar
- 47 Forbes CD, Toth JG, Ozbal CC et al. High-throughput mass spectrometry screening for inhibitors of phosphatidylserine decarboxylase. J. Biomol. Screen.12(5),628–634 (2007).Crossref, Medline, CAS, Google Scholar
- 48 Soulard P, McLaughlin M, Stevens J et al. Development of a high-throughput screening assay for stearoyl-coa desaturase using rat liver microsomes, deuterium labeled stearoyl-coa and mass spectrometry. Analytica Chimica Acta.627(1),105–111 (2008).Crossref, Medline, CAS, Google Scholar
- 49 Jonas M, LaMarr WA, Ozbal C. Mass spectrometry in high-throughput screening: a case study on acetyl-coenzyme a carboxylase using RapidFire – mass spectrometry (RF-MS). Comb. Chem. High Throughput Screen.12(8),752–759 (2009).Crossref, Medline, CAS, Google Scholar
- 50 Lim KB, Ozbal CC, Kassel DB. Development of a high-throughput online solid-phase extraction/tandem mass spectrometry method for cytochrome P450 inhibition screening. J. Biomol. Screen.15(4),447–452 (2010).Crossref, Medline, CAS, Google Scholar
- 51 Motlekar N, Diamond SL, Napper AD. Evaluation of an orthogonal pooling strategy for rapid high-throughput screening of proteases. Assay Drug Dev. Technol.6(3),395–405 (2008).Crossref, Medline, CAS, Google Scholar
- 52 Langsdorf EF, Malikzay A, Lamarr WA et al. Screening for antibacterial inhibitors of the UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) using a high-throughput mass spectrometry assay. J. Biomol. Screen.15(1),52–61 (2010).Crossref, Medline, CAS, Google Scholar
- 53 Cummings MD, Farnum MA, Nelen MI. Universal screening methods and applications of ThermoFluor. J. Biomol. Screen.11(7),854–863 (2006).Crossref, Medline, CAS, Google Scholar
- 54 Clare JJ. Targeting ion channels for drug discovery. Discov. Med.9(46),253–260 (2010).Medline, Google Scholar
- 55 Milligan G, Kostenis E. Heterotrimeric G-proteins: a short history. Br. J. Pharmacol.147(Suppl. 1),S46–S55 (2006).Crossref, Medline, CAS, Google Scholar
- 56 Mailman RB. GPCR functional selectivity has therapeutic impact. Trends Pharmacol. Sci.28(8),390–396 (2007).Crossref, Medline, CAS, Google Scholar
- 57 Zheng H, Loh HH, Law PY. Agonist-selective signaling of G protein-coupled receptor: mechanisms and implications. IUBMB Life62(2),112–119 (2010).Medline, CAS, Google Scholar
- 58 Prather PL, Martin NA, Breivogel CS, Childers SR. Activation of cannabinoid receptors in rat brain by WIN 55212–2 produces coupling to multiple G protein α-subunits with different potencies. Mol. Pharmacol.57(5),1000–1010 (2000).Medline, CAS, Google Scholar
- 59 Houston DB, Howlett AC. Differential receptor-G-protein coupling evoked by dissimilar cannabinoid receptor agonists. Cell Signal.10(9),667–674 (1998).Crossref, Medline, CAS, Google Scholar
- 60 Rajagopal S, Rajagopal K, Lefkowitz RJ. Teaching old receptors new tricks: biasing seven-transmembrane receptors. Nat. Rev. Drug Discov.9(5),373–386 (2010).▪▪ Discusses the roles of biased ligands in receptor bioactivity and as therapeutics.Crossref, Medline, CAS, Google Scholar
- 61 Ferré S, Goldberg SR, Lluis C, Franco R. Looking for the role of cannabinoid receptor heteromers in striatal function. Neuropharmacology56(Suppl 1),226–234 (2009).Crossref, Medline, CAS, Google Scholar
- 62 Sexton PM, Poyner DR, Simms J, Christopoulos A, Hay DL. Modulating receptor function through RAMPs: can they represent drug targets in themselves? Drug Discov. Today.14(7–8),413–419 (2009).Crossref, Medline, CAS, Google Scholar
- 63 Traynor J. Regulator of G protein-signaling proteins and addictive drugs. Ann. NY Acad. Sci.1187,341–352 (2010).Crossref, Medline, CAS, Google Scholar
- 64 Maudsley S, Martin B, Luttrell LM. G protein-coupled receptor signaling complexity in neuronal tissue: implications for novel therapeutics. Curr. Alzheimer Res.4(1),3–19 (2007).Crossref, Medline, CAS, Google Scholar
- 65 Galandrin S, Oligny-Longpré G, Bouvier M. The evasive nature of drug efficacy: implications for drug discovery. Trends Pharmacol. Sci.28(8),423–430 (2007).▪ Discusses the impact of complex receptor biology on the drug discovery process.Crossref, Medline, CAS, Google Scholar
- 66 Banerjee P, Franz B, Bhunia AK. Mammalian cell-based sensor system. Adv. Biochem. Eng. Biotechnol.117,21–55 (2010).Medline, CAS, Google Scholar
- 67 Cabral GA, Griffin-Thomas L. Emerging role of the cannabinoid receptor CB2 in immune regulation: therapeutic prospects for neuroinflammation. Expert Rev. Mol. Med.11,e3 (2009).Crossref, Medline, Google Scholar
- 68 Idris AI, van ‘t Hof RJ, Greig IR et al. Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors. Nat. Med.11(7),774–779 (2005).Crossref, Medline, CAS, Google Scholar
- 69 Idris AI, Sophocleous A, Landao-Bassonga E, van’t Hof RJ, Ralston SH. Regulation of bone mass, osteoclast function and ovariectomy-induced bone loss by the type 2 cannabinoid receptor. Endocrinology.149(11),5619–5626 (2008).Crossref, Medline, CAS, Google Scholar
- 70 Ofek O, Karsak M, Leclerc N et al. Peripheral cannabinoid receptor, CB2, regulates bone mass. Proc. Natl Acad. Sci. USA103(3),696–701 (2006).Crossref, Medline, CAS, Google Scholar
- 71 Lunn CA, Fine J, Rojas-Triana A et al. Cannabinoid CB(2)-selective inverse agonist protects against antigen-induced bone loss. Immunopharmacol. Immunotoxicol.29(3–4),387–401 (2007).Crossref, Medline, CAS, Google Scholar
- 72 Lozano-Ondoua AN, Wright C, Vardanyan A et al. A cannabinoid 2 receptor agonist attenuates bone cancer-induced pain and bone loss. Life Sci.86(17–18),646–653 (2010).Crossref, Medline, CAS, Google Scholar
- 73 Geng D, Xu Y, Yang H et al. Protection against titanium particle induced osteolysis by cannabinoid receptor 2 selective antagonist. Biomaterials31(8),1996–2000 (2010).Crossref, Medline, CAS, Google Scholar
- 74 Ni X, Geller EB, Eppihimer MJ, Eisenstein TK, Adler MW, Tuma RF. Win 55212–2, a cannabinoid receptor agonist, attenuates leukocyte/endothelial interactions in an experimental autoimmune encephalomyelitis model. Mult. Scler.10(2),158–164 (2004).Crossref, Medline, CAS, Google Scholar
- 75 Maresz K, Pryce G, Ponomarev ED et al. Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells. Nat. Med.13(4),492–497 (2007).Crossref, Medline, CAS, Google Scholar
- 76 Sipe JC, Arbour N, Gerber A, Beutler E. Reduced endocannabinoid immune modulation by a common cannabinoid 2 (CB2) receptor gene polymorphism: possible risk for autoimmune disorders. J. Leukoc. Biol.78(1),231–238 (2005).Crossref, Medline, CAS, Google Scholar
- 77 Palazuelos J, Davoust N, Julien B et al. The CB(2) cannabinoid receptor controls myeloid progenitor trafficking: involvement in the pathogenesis of an animal model of multiple sclerosis. J. Biol. Chem.283(19),13320–13329 (2008).Crossref, Medline, CAS, Google Scholar
- 78 Lunn CA, Reich EP, Fine JS et al. Biology and therapeutic potential of cannabinoid CB2 receptor inverse agonists. Br. J. Pharmacol.153(2),226–239 (2008).Crossref, Medline, CAS, Google Scholar
- 79 Lunn CA. Updating the chemistry and biology of cannabinoid CB(2) receptor-specific inverse agonists. Curr. Top. Med. Chem.10(8),768–778 (2010).Crossref, Medline, CAS, Google Scholar
- 80 Cotton M, Claing A. G protein-coupled receptors stimulation and the control of cell migration. Cell Signal.21(7),1045–1053 (2009).▪▪ Describes downstream effects of receptor activation on cell migration, with a focus on receptor bioactivity.Crossref, Medline, CAS, Google Scholar
- 81 DeFea K. β-arrestins and heterotrimeric G-proteins: collaborators and competitors in signal transduction. Br. J. Pharmacol.153(Suppl. 1),S298–S309 (2008).▪▪ Describes downstream effects of receptor activation on cell migration, with focus on contribution of β-arrestins.Crossref, Medline, CAS, Google Scholar
- 82 Ganguly S, Pucadyil TJ, Chattopadhyay A. Actin cytoskeleton-dependent dynamics of the human serotonin1A receptor correlates with receptor signaling. Biophys. J.95(1),451–463 (2008).Crossref, Medline, CAS, Google Scholar
- 83 Johnson EN. High throughput screening follow-up studies using physiologically relevant cells. Am. Drug Discov.3(2),12–22 (2007).Google Scholar
- 84 Cunningham BT, Li P, Schulz S et al. Label-free assays on the BIND system. J. Biomol. Screen.9(6),481–490 (2004).Crossref, Medline, CAS, Google Scholar
- 85 Fang Y, Ferrie AM, Li G. Probing cytoskeleton modulation by optical biosensors. FEBS Lett.579(19),4175–4180 (2005).Crossref, Medline, CAS, Google Scholar
- 86 Verdonk E, Johnson K, McGuinness R et al. Cellular dielectric spectroscopy: a label-free comprehensive platform for functional evaluation of endogenous receptors. Assay Drug Dev. Technol.4(5),609–619 (2006).Crossref, Medline, CAS, Google Scholar
- 87 Solly K, Wang X, Xu X, Strulovici B, Zheng W. Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays. Assay Drug Dev. Technol.2(4),363–372 (2004).Crossref, Medline, CAS, Google Scholar
- 88 Lee PH, Gao A, van Staden C et al. Evaluation of dynamic mass redistribution technology for pharmacological studies of recombinant and endogenously expressed G protein-coupled receptors. Assay Drug Dev.Technol.6,83–94 (2008).Crossref, Medline, Google Scholar
- 89 Yu N, Atienza JM, Bernard J et al. Real-time monitoring of morphological changes in living cells by electronic cell sensor arrays: an approach to study G protein-coupled receptors. Anal. Chem.78,35–43 (2006).Crossref, Medline, CAS, Google Scholar
- 90 Peters MF, Knappenberger KS, Wilkins D et al. Evaluation of cellular dielectric spectroscopy, a whole-cell, label-free technology for drug discovery on Gi-coupled GPCRs. J. Biomol. Screen.12,312–319 (2007).Crossref, Medline, CAS, Google Scholar
- 91 Peters MF, Scott CW. Evaluating cellular impedance assays for detection of GPCR pleiotropic signaling and functional selectivity. J. Biomol. Screen.14,246–255 (2009).Crossref, Medline, CAS, Google Scholar
- 92 Peters MF, Vaillancourt F, Heroux M, Valiquette M, Scott CW. Comparing label-free biosensors for pharmacological screening with cell-based functional assays. Assay Drug Dev. Technol.8(2),219–227 (2010).▪ Investigators provide direct comparison of multiple label-free biosensor platforms.Crossref, Medline, CAS, Google Scholar
- 93 Du Y, Li Z, Li L et al. Distinct growth factor-induced dynamic mass redistribution (DMR) profiles for monitoring oncogenic signaling pathways in various cancer cells. J. Recept. Signal. Trans.29(3–4),182–194 (2009).Crossref, Medline, CAS, Google Scholar
- 94 Fang Y, Ferrie AM. Label-free optical biosensor for ligand-directed functional selectivity acting on β(2) adrenoceptor in living cells. FEBS Lett.582(5),558–564 (2008).Crossref, Medline, CAS, Google Scholar
- 95 Dodgson K, Gedge L, Murray DC, Coldwell M. A 100K well screen for a muscarinic receptor using the Epic® label-free system – a reflection on the benefits of the label-free approach to screening seven-transmembrane receptors. J. Recept. Signal Trans.29(3–4),163–172 (2009).▪▪ Investigator provides direct evidence of the potential benefits of label-free biosensors as a screening tool for seven transmembrane receptor targets.Crossref, Medline, CAS, Google Scholar
- 96 Begley S, Carmichael M. Desperately seeking cures. Newsweek. May 24 & 31, 38–42 (2010).Google Scholar
- 97 Rathore R, Pribil P, Corr JJ, Seibel WL, Evdokimov A, Greis KD. Multiplex enzyme assays and inhibitor screening by mass spectrometry. J. Biomol. Screen.15(8),1001–1007 (2010) (Epub).Crossref, Medline, CAS, Google Scholar
- 98 Bowen BP, Northen TR. Dealing with the unknown: metabolomics and metabolite atlases. J. Am. Soc. Mass. Spectrom.21(9),1471–1476 (2010).Crossref, Medline, CAS, Google Scholar
- 99 Young NL, Plazas-Mayorca MD, Garcia BA. Systems-wide proteomic characterization of combinatorial post-translational modification patterns. Expert Rev. Proteomics.7(1),79–92 (2010).Crossref, Medline, CAS, Google Scholar
- 100 Wang D, Bodovitz S. Single cell analysis: the new frontier in ‘omics’. Trends Biotechnol.28(6),281–290 (2010). (Epub).Crossref, Medline, CAS, Google Scholar
- 101 Baxter MA, Rowe C, Alder J et al. Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening. Stem Cell Res.5(1),4–22 (2010).Crossref, Medline, CAS, Google Scholar
- 102 Murata M, Tohyama S, Fukuda K. Impacts of recent advances in cardiovascular regenerative medicine on clinical therapies and drug discovery. Pharmacol. Ther.126(2),109–118 (2010).Crossref, Medline, CAS, Google Scholar
- 103 Phillips BW, Crook JM. Pluripotent human stem cells: a novel tool in drug discovery. BioDrugs24(2),99–108 (2010).Crossref, Medline, CAS, Google Scholar
- 104 Sano Y, Shimizu F, Abe M et al. Establishment of a new conditionally immortalized human brain microvascular endothelial cell line retaining an in vivo blood–brain barrier function. J. Cell. Physiol.225(2),519–528 (2010).Crossref, Medline, CAS, Google Scholar
- 105 Gu Y, Li H, Miki J et al. Phenotypic characterization of telomerase-immortalized primary non-malignant and malignant tumor-derived human prostate epithelial cell lines. Exp. Cell Res.312(6),831–843 (2006).Crossref, Medline, CAS, Google Scholar
- 106 Brandt S. TERT over-expression affects the growth of myocardial tissue derived from mouse embryonic stem cells. Differentiation,79(1),1–8 (2010).Crossref, Medline, CAS, Google Scholar
- 107 Cheung PY, Deng W, Man C et al. Genetic alterations in a telomerase-immortalized human esophageal epithelial cell line: implications for carcinogenesis. Cancer Lett.293(1),41–51 (2010).Crossref, Medline, CAS, Google Scholar
- 108 SRU Biosystems. Introducing the BIND® Scanner: low cell number applications in cytotoxicity, chemotaxis and differentiation. Presented at: SBS 16th Annual Conference & Exhibition, Phoenix, AZ, USA, 13 April 2010.Google Scholar
- 109 Huang WE, Li M, Jarvis RM, Goodacre R, Banwart SA. Shining light on the microbial world the application of Raman microspectroscopy. Adv. Appl. Microbiol.70,153–186 (2010).Crossref, Medline, CAS, Google Scholar
- 110 Wachsmann-Hogiu S, Weeks T, Huser T. Chemical analysis in vivo and in vitro by Raman spectroscopy – from single cells to humans. Curr. Opin. Biotechnol.20(1),63–73 (2009).Crossref, Medline, CAS, Google Scholar
- 111 Bleicher KH, Böhm HJ, Müller K, Alanine AI. Hit and lead generation: beyond high-throughput screening. Nat. Rev. Drug Discov.2(5),369–378 (2003).Crossref, Medline, CAS, Google Scholar
- 112 Keseru GM, Makara GM. Hit discovery and hit-to-lead approaches. Drug Discov. Today11(15–16),741–748 (2006).Crossref, Medline, Google Scholar
- 113 Feng BY, Shoichet BK. Synergy and antagonism of promiscuous inhibition in multiple-compound mixtures. J. Med. Chem.49(7),2151–2154 (2006).Crossref, Medline, CAS, Google Scholar
- 114 Jadhav A, Ferreira RS, Klumpp C et al. Quantitative analyses of aggregation, autofluorescence and reactivity artifacts in a screen for inhibitors of a thiol protease. J. Med. Chem.53(1),37–51 (2010).Crossref, Medline, CAS, Google Scholar
- 201 Duncan DE. Pharma and biotech: is there love? Fortune, 10 May 2010. http://money.cnn.com/2010/05/10/news/companies/pharma_biotech_marriage.fortune/Google Scholar
- 202 Herper M. Why Glaxo Bought Sirtris. FORBES, April 24 (2008) www.forbes.com/2008/04/23/pharmacuticals-sirtris-glaxosmithkline-biz-healthcare-cx_mh_0424glaxo.htmlGoogle Scholar

