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To date there has been a considerable amount of interest andsuccess in the pharmaceutical industry in the discovery of drugtargets and diagnostics via genomic technologies, namely DNAsequencing, mutation/polymorphism detection and expressionmonitoring of mRNA. As the ultimate targets for the majority ofthese methods are actually proteins, more and more emphasishas been placed upon protein-based methods in an effort todefine the function of proteins discovered by genomictechnologies. One of the most challenging areas of drug targetdiscovery facing researchers today is the search for novelreceptor–ligand pairs. Database mining techniques inconjunction with other computational methods are able toidentify many novel sequences of putative receptors, but theability to similarly identify the receptor’s natural ligand is notpossible by these methods. The past few years have seen anincrease in methodology and instrumentation focused on theability to discover and characterize protein–protein interactions,as well as receptor–ligand pairs. Significant advances havebeen made in the areas of instrumentation (biosensors andfluorescent plate readers) as well as methodologies relating tophage/ribosome display and library construction.

AddressMillennium Pharmaceuticals, 640 Memorial Drive, Cambridge,MA 02139-4853, USA

Current Opinion in Biotechnology 2000, 11:42–46

0958-1669/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved.

AbbreviationsFLIPR fluorometric imaging plate readerGPCR G-protein-coupled receptorSPR surface plasmon resonance

IntroductionThere are many receptors of various predicted biologicalfunctions that have no known ligand; such receptors arecommonly referred to as orphans. Ligand fishing or de-orphaning is the process by which these receptors arescreened against a multitude of compounds or cell/tissueextracts to identify putative ligands for the receptor. Thisprocess is not limited to orphan receptors and ligands.Similar techniques can be applied to novel proteins thathave no known binding partner. The type of assay and thesource of the ligand library are factors that should be care-fully considered before the process begins. Receptors canbe screened for ligands that simply bind to the receptor, orfor ligands that activate the receptor. Receptor activation isusually assessed by an in vivo assay that can quantitativelymeasure the production of certain second messenger mole-cules, such as calcium, or changes in pH. These criteria arealso indicative of the classes of technologies used in ligandfishing. There are specific analytical techniques that are

used to identify and characterize protein binders as well astechniques that are specific for the measurement of recep-tor activation. This review will outline advances in thesetechniques and the following sections will focus on exam-ples of each type of methodology, that is, screens to detectdirect binding and screens that detect receptor activation.

Screens that detect direct bindingThe most convenient methodologies for ligand fishing arethose based on direct binding. As stated previously, thesetypes of assays are not assessed by the ligands ability toactivate a receptor or enzyme. A common configuration ofthese types of tests consists of the target protein immobi-lized on a solid support, the source of possible ligand ismixed with or passed over the immobilized target proteinand after a washing step(s) the putative ligand is eluted(e.g. by pH change or salt washes) and characterized bysome other analytical method. The most common form ofthis analysis is immunoprecipitation [1]. Drawbacks of thistype of analysis include the possibility of non-specificbinding and a general lack of sensitivity. There are a num-ber of new analytical techniques being developed toaddress these limitations. The following two technologieswill make significant improvements in the area of directbinding assays.

Surface plasmon resonance biosensors Recent advances in analytical instrumentation, includinganalytical ultracentrifugation, bomb calorimetry andbiosensor technology [2,3], have had a great impact on theability to detect and measure biospecific interactions inreal time. One of the most versatile of these techniques isbiosensors based on the optical phenomenon of surfaceplasmon resonance (SPR). The SPR phenomenon hasbeen known for over 25 years and the theory is fairly welldeveloped [4]. A surface plasmon is an oscillation of freeelectrons that propagates along the surface of a thin film ofmetal, such as silver or gold. Energy carried by photons oflight can be ‘coupled’ or transferred to these free electronsin the metal. When there is a match, or resonance, betweenthe energy of the light photons and the energy of the elec-trons at the metal surface, coupling or energy transferoccurs. The coupling can be observed by measuring theamount of light reflected by the metal surface. All the lightat most wavelengths is reflected except at the resonantwavelength, where almost all the light is absorbed. Achemical change at the surface results in a shift in thewavelength of light that is absorbed rather than reflected(resonant wavelength), and the magnitude of that shift isquantitatively related to the magnitude of the chemicalchange. The chemical change that is being measured inmost SPR-based biosensors is caused by an increase inmass close to the metal surface.

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Biotechnology match making: screening orphan ligands and receptors Williams 43

A common configuration of biosensors is the use of contin-uous flow technology to monitor interactions. One of theinteractants is immobilized on the sensor surface and theother interactant flows continuously over the sensor sur-face. As more and more molecules from the solution bindto the immobilized interactant, the resonant wavelengthchanges and a response is registered. The amount bywhich the wavelength changes is proportional to theamount of ligand that binds, thus giving a quantitativereading. The ability to detect and quantify biospecificinteractions from complex fluids, cell lysates, conditionedmedia and a variety of other sources makes biosensors alogical method for orphan ligand screening. Applicationsusing this technology for ligand fishing have been pub-lished recently [5–7]. Biosensors are important tools forligand screening because they are very versatile. The ver-satility arises from the fact that the measurements can bemade quickly in real time, the interactants usually requirelittle if any chemical modification and very little material isconsumed. One important drawback of this methodologyis that once a sample is identified as having a possible lig-and, classical biochemical techniques need to be applied toidentify the new ligand. This requires liters of cell cultureproduction and sometimes-complicated high performanceliquid chromatography methods development. In the pastfew years, there has been an increased interest in the abil-ity to identify the bound protein(s) directly from the chipsurface. The use of sensitive mass spectrometers andadvanced database searching algorithms has made thisanalysis possible [8••,9–11] (Figure 1).

Directed protein evolutionThe process of directed in vitro evolution using nucleicacids by combining rounds of mutation and selective pres-sure has been utilized to select nucleic acid molecules witha wide variety of affinities and catalytic properties [12–15].Because proteins are the predominant molecules used as

diagnostic and therapeutic reagents it would be of greatvalue to develop methods for the in vitro selection and evo-lution of proteins. The first methods that succeeded in thisarea, such as phage display [16] and plasmid display[17,18], involved the in vivo transcription and translation ofDNA/mRNA in a suitable host, usually yeast or Escherichiacoli. The resulting proteins were linked to the encodingnucleic acid, which could be recovered, for amplificationand further testing. During amplification rounds manymutations can be made, thus allowing screening forstronger or weaker binders. A key drawback in thismethodology arose from limitations in transfecton efficien-cy, which limits the diversity of proteins expressed to lessthan 109. Two relatively recent advances have taken thisapproach to a total in vitro environment. The basis for bothtechniques is ribosome display [19–22], in which the newlyformed peptide is displayed on the ribosome that is trans-lating it. This method, in contrast to the earlier techniques,is totally in vitro and has the potential for the constructionof libraries with a complexity of as many as 1012 elements.Ribosome display requires that the mRNA and resultingpeptide stay in a complex with the ribosome in order tomaintain the association of the selected protein with theencoding nucleic acid for further analysis. A recentadvance in this method [23,24••] uses a tRNA analog,puromycin, to covalently link the nascent peptide to themRNA, forming a mRNA–peptide fusion that is very sta-ble (Figure 2). This technology has a very promising futureas a source of peptide or protein molecules for ligandscreening. Experiments could be designed in which alibrary of 1012 or greater elements is synthesized and usedin a screen of an orphan receptor. Other possible uses arefor novel substrate identification for various kinases or pro-teases. The most powerful aspect of this method is the factthat the nucleic acid information that was used to synthe-size the protein molecule is linked directly to themolecule. Upon isolation of active fusions, amplification of

Figure 1

An example of the use of biosensortechnology coupled with mass spectrometryfor ligand identification. (a) Binding analysis ofligand binding to immobilized membranes on asensor chip surface. This shows a sensorgramtrace of peptide binding to immobilizedGPCRs. The bound ligand is eluted from thechip and analyzed by mass spectrometry.(b) Liquid chromatograph of eluted peptidefrom biosensor. The sum peak is the peak ofall the mass spectra that comprise the signaland shows that there is a specific peptidebeing analyzed. (c) The mass spectra thatcomprise the sum peak are shown individuallyand show the same peptide in multiple chargestates.

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the nucleic acid sequence and sequencing of the resultingproduct can be used to identify the protein. RNA–peptidefusions have the potential to yield a staggering diversity ofdirected libraries that could be used in the search for novelligands or protein interactors.

Screens that detect receptor activationWhen probing protein receptor interactions, a key proper-ty of any ligand is its ability to elicit a specific cellularresponse. Direct binding assays are limited in that they donot discriminate between agonists and antagonists.Receptors expressed in cells that enable the receptor to beexpressed in an active state and also to be functionallylinked to a signal transduction pathway will give the mostvaluable information about putative ligands.

Cytosensor microphysiometerMany receptors can couple to more than one signal trans-duction pathway. Most of the current assay technologiesdo not allow for the characterization of the mechanism bywhich receptor activation effects or regulates cellularphysiology. Cytosensor microphysiometers offer a way ofmeasuring cellular responses by detecting minute changesin extracellular acidification [25,26]. Energy metabolism isprimarily coupled to cellular ATP usage. Cellular ATPlevels fluctuate upon receptor activation and initiation ofsignal transduction and increased ATP production causesthe amount of H+ secreted by the cell to increase, thuscausing a change in acid excretion to the surrounding

media. Receptor activation may also affect acidification byaltering the Na+/H+ exchange. The cytosensor microphys-iometer uses a silicon sensor to detect cellular responses toa wide variety of effector agents. Cells expressing a recep-tor of interest are placed between two porous membranesinside disposable capsules, which are continually washedwith a flow of culture medium. Putative ligands of inter-est can be introduced into the chamber and incubated.Modifications in the metabolic activity due to receptoractivation result in changes in the rate of excretion ofacidic products, which can be detected by the instrumentas a change in pH. The cytosensor microphysiometrysystem can quantitate or create membrane receptor-medi-ated response profiles of viable cells to definedconcentrations of ligands. These ligands could includeagonists and antagonists of neurotransmitters, hormone,cytokine and growth factor receptors, and avariety of other proteins and peptides interactors.Microphysiometers are a powerful technology for thecharacterization of receptor signaling pathways [27•],receptor subtype classification (e.g. by ligands that bindthem or cells they are expressed in) [28,29], and is fastbecoming a more valuable tool for ligand fishing [30••].The cytosensor microphysiometer, though extremely sen-sitive, is not a high-throughput instrument. Incubationtimes and washing steps between samples can be quitelong. Certain commercial interests have claimed higher-throughput microphysiometers but they have yet tobecome widely available.

44 Analytical biotechnology

Figure 2

A schematic diagram showing the process ofRNA–protein fusion synthesis. MessengerRNA created with the puromycin adaptoranalog is translated in vitro. The nascentpeptide chain is covalently attached to theadaptor analog via an amide linkage. Adaptedfrom [23] with permission.

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Fluorometric imaging plate readerThe fluorometric imaging plate reader (FLIPR) system isa highly advanced plate reader that is able to performhomogeneous, kinetic, and cell-based fluorometric assays,such as the measurement of intracellular calcium concen-tration, membrane potential and intracellular pH. Thesystem utilizes optics, fluidics and temperature control tosimultaneously stimulate and optically read all 96 wells ofa microplate within one second. The system works with avariety of cell types that can be both adherent and non-adherent. The most extensive use of the FLIPR system todate has been in measuring intracellular calcium concen-tration [30••]. Intracellular calcium mobilization is animportant indicator of receptor activation for many recep-tor types, but especially so for the G-protein-coupledreceptors (GPCRs). The GPCRs have been described asamong the largest families of genes in the genome [31] andare one of the most therapeutically relevant receptor class-es today [32•,33••]. For example, angiotensin receptors areGPCRs and many cardiovascular therapeutics are targetedat GPCRs. A target cell line can be transfected with thereceptor of interest along with the appropriate G-proteinsubunit [34•] to create a functional signaling system. Theresulting cell lines can be exposed to peptides, proteins orsmall molecules that could potentially activate the recep-tor [35]. The FLIPR system has the advantage of being ahigh-throughput system with the rapid acquisition of 96 or384 wells of data. The FLIPR system is fast becoming themethod of choice for many laboratories in their efforts forde-orphaning GPCRs.

ConclusionsThe review of the techniques and methodologies outlinedhere is by no means comprehensive, but a quick look at afew of the most interesting and promising methods foridentifying novel protein–protein interactions. There areadvantages to both types of systems presented, directbinding and receptor activation. Direct binding assayshave the advantage of speed and the possibility fortremendous diversity in interacting molecules. Directbinding assays have the disadvantages of not measuringreceptor activation and the inability to distinguish betweenagonists and antagonists. Cellular activation assays revealmuch more information about the ligand–receptor interac-tion, including the possibility of deriving kineticparameters, receptor deactivation, and signal transductionpathway elucidation. What does the future hold for ligandfishing or de-orphaning? Continued refinement of currentsystems should dramatically increase the throughput capa-bilities of cell-based screens. The configurations of directbinding assays are continually getting smaller, it is entirelypossible that within the next few years protein arrays willbe seen with similar densities to the DNA arrays of today.Finally, there is an effort to map protein function and pro-tein–protein interactions by computational methods [36•].This would be accomplished by scanning all availabledatabases of DNA sequence and comparing the sequencesof known protein pairs with the homologs of the same

proteins in other species, thus creating an in silico map ofprotein–protein interactions.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Coligan JE, Kruisbeck AM, Margulies DH, Shevach EH, Strober W:Current Protocols in Immunology. New York: John Wiley and Sons;1999.

2. Teller DC: Characterization of proteins by sedimentationequilibrium in the analytical ultracentrifuge. Methods Enzymol1973, 27:346-441.

3. Martin CJ, Marini MA: Micro calorimetry in biochemical analysis.Crit Rev Anal Chem 1979, 8:221.

4. Kretschmann E, Raether H: Radiative decay of non-radiativesurface plasmons excited by light. Naturforschung 1968,A23:2135-2136.

5. Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, Jain V, Ryan TE,Bruno J, Radziejewski C, Maisonpierre PC, Yancopoulos GD:Isolation of angiopoietin-1, a ligand for the TIE2 receptor, bysecretion-trap expression cloning. Cell 1996, 87:1161-1169.

6. Sakano S, Serizawa R, Inada T, Iwama A, Itoh A, Kato C, Shimizu Y,Shinkai F, Kondo S, Ohno M, Suda T: Characterization of a ligandfor receptor protein tyrosine kinase HTK expressed in immaturehemopoietic cells. Oncogene 1996, 13:813-822.

7. Lackmann M, Bucci T, Mann RJ, Kraveks LA, Viney E, Smith F, Moritz RL,Carter W, Simpson RJ, Nicola NA: Purification of the EPH-likereceptor HEK using a biosensor-based affinity detectionapproach. Proc Natl Acad Sci USA 1996, 93:2523-2527.

8. Williams C, Addona TA: The integration of SPR biosensors with •• mass spectrometry: possible applications for proteome analysis.

Trends Biotechnol 2000, in press.An excellent review of current integration strategies for SPR-based biosen-sors.

9. Nelson RW, Krone JR, Osten J: Surface plasmon resonancebiomolecular interaction analysis mass spectrometry. 1. Chip-based analysis. Anal Chem 1997, 69:4363-4368.

10. Roepstorff P: Mass spectrometry in protein studies from genometo function. Curr Opin Biotechnol 1997, 8:6-13.

11. Nelson RW, Krone JR, Osten J: Surface plasmon resonancebiomolecular interaction analysis mass spectrometry. 2. Fiberoptic-based analysis. Anal Chem 1997, 69:4369-4374.

12. Gold L, Polisky B, Uhlenbeck O, Yarus M: Diversity ofoligonucleotide functions. Annu Rev Biochem 1995, 64:763-797.

13. Irvine D, Tuerk C, Gold L: SELEXION. Systematic evolution ofligands by exponential enrichment with integrated optimization bynon-linear analysis. J Mol Biol 1991, 222:739-761.

14. Beaudry AA, Joyce GF: Directed evolution of an RNA enzyme.Science 1992, 257:635-641.

15. Bartel DP, Szostak JW: Isolation of new ribozymes from a largepool of random sequences. Science 1993, 26:1411-1418.

16. Smith GP: Filamentous fusion phage: novel expression vectorsthat display cloned antigens on the virion surface. Science 1985,228:1315-1317.

17. Schatz PJ, Cull MG, Martin EL, Gates CM: Screening of peptidelibraries linked to lac repressor. Methods Enzymol 1996, 267:171-191.

18. Gates CM, Stemmer WP, Kaptein R, Schatz PJ: Affinity selectiveisolation of ligands from peptide libraries through display on a lacrepressor ‘headpiece dimer’. J Mol Biol 1996, 255:373-386.

19. Mattheakis LC, Bhatt RR, Dower WJ: An in vitro polysome displaysystem for identifying ligands from very large peptide libraries.Proc Natl Acad Sci USA 1994, 91:9022-9026.

20. Mattheakis LC, Dias JM, Dower WJ: Cell-free synthesis of peptidelibraries displayed on polysomes. Methods Enzymol 1996,267:195-207.

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21. Hanes J, Plückthun A: In vitro selection and evolution of functionalproteins by using ribosome display. Proc Natl Acad Sci USA 1997,94:4937-4942.

22. Hanes J, Jermutus L, Schaffitzel C, Pluckthun A: Comparison ofEscherichia coli and rabbit reticulocyte ribosome display systems.FEBS Lett 1999, 450:105-110.

23. Roberts RW, Szostak JW: RNA–peptide fusions for the in vitroselection of peptides and proteins. Proc Natl Acad Sci USA 1997,94:12297-12302.

24. Roberts RW: Totally in vitro protein selection using mRNA–protein •• fusions and ribosome display. Curr Opin Chem Biol 1999,

3:268-273.A comprehensive review of methodologies used in mRNA–protein fusionand ribosome display.

25. Owicki JC, Parce JW: Biosensors based on the energymetabolism of living cells: the physical chemistry and cell biologyof extracellular acidification. Biosens Bioelectron 1992, 7:255-272.

26. McConnell HM, Owicki JC, Parce JW, Miller DL, Baxter GT, Wada HG,Pitchford S: The cytosensor microphysiometer: biologicalapplications of silicon technology. Science 1992, 257:1906-1912.

27. Chen L, Tashjian AH: Identification of distinct signalling pathways • for somatostatin receptors SSTR1 and SSTR2 as revealed by

microphysiometry. Cell Signal 1999, 11:499-505.An interesting paper on the use of microphysiometry and pathway/ligandcharacterization.

28. Coldwell MC, Boyfield I, Brown AM, Stemp G, Middlemiss DN:Pharmacological characterization of extracellular acidification rateresponses in human D2 (long), D3 and D4.4 receptors expressedin Chinese hamster ovary cells. Br J Pharmacol 1999, 127:1135-1144.

29. Starback P, Lundell I, Fredriksson R, Berglund MM, Yan YL, Wraith A,Söderberg C, Postlethwait JH, Larhammar D: Neuropeptide Yreceptor subtype with unique properties cloned in the zebrafish:the zYa receptor. Brain Res Mol Brain Res 1999, 70:242-252.

30. Sullivan E, Tucker EM, Dale IL: Measurement of [Ca2+] using the •• Fluorometric Imaging Plate Reader (FLIPR). Methods Mol Biol

1999, 114:125-133.Rationale on the use of FLIPR as a screening tool.

31. Brann MR, Messier T, Dorman C, Lannigan D: Cell-based assays forG-protein-coupled/tyrosine kinase-coupled receptors. J BiomolScreening 1996, 1:43-45.

32. Marchese A, George SR, Kolakowski LF Jr, Lynch KR, O’Dowd BF: • Novel GPCR’s and their endogenous ligands: expanding the

boundaries of physiology and pharmacology. Trends PharmacolSci 1999, 20:370-375.

This paper details the pharmacological implications of G-protein-coupledreceptors in research.

33. Wilson S, Bergsma DJ, Chambers JK, Muir AL, Fantom KG, Ellis C, •• Murdock PR, Herrity NC, Stadel JM: Orphan G-protein-coupled

receptors: the next generation of drug targets? Br J Pharmacol1998, 125:1387-1392.

An outstanding review on the importance of G-coupled-protein receptors incurrent drug discovery.

34. Coward P, Chan SD, Wada HG, Humphries GM, Conklin BR: • Chimeric G proteins allow a high-throughput signaling assay of

Gi-coupled receptors. Anal Biochem 1999, 270:242-248.The use of chimeric G-proteins in high-throughput screening is described.

35. Cao J, O’Donnell D, Vu H, Payza K, Pou C, Godbout C, Jakob A,Pelletier M, Lembo P, Ahmad S, Walker P: Cloning andcharacterization of a cDNA encoding a novel subtype of ratthyrotropin-releasing hormone receptor. J Biol Chem 1998,273:32281-32287.

36. Marcotte EM, Pellegrini M, Ng HL, Rice DW, Yeates TO, Eisenberg D: • Detecting protein function and protein-protein interactions from

genome sequences. Science 1999, 285:751-753.This paper outlines a computational method for predicting protein–proteininteractors using cross species homologies.

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