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Fluorescent-protein biosensors: new tools for

drug discovery Kenneth A. Giuliano and D. Lansing Taylor

Recent improvements in target discovery and high-throughput screening have

increased the pressure at key points along the drug-discovery pipeline. High-content

screening was developed to ease the bottlenecks formed at the target-validation and

lead-optimization points, and a new generation of reagents that report on specific

molecular processes in li~ing cells (fluorescent-protein biosensors) have been

important in its development. Creative designs of fluorescent-protein biosensors have

emerged and been used to measure the molecular dynamics of macromolecules,

metabolites and ions. Recent applications of fluorescent-protein biosensors to

biological problems have provided a foundation for their use in biotechnology.

The challenge for the biological and biotechnological sciences today is to decipher the 'language' of the living cell. Components of this language, the myriad molecular reactions that take place in each cell, are only beginning to be interpreted. The complete one-dimensional sequence of the humarL genome is within reach, and genomics has already had an impact on our understanding of the genetic basis of the nor- mal and diseased states of cells and organisms. Pro- teomics, the process of coupling macromolecular struc- ture with exquisitely specific function, is continuously being used to link genomics and protein-structural and -fnnctional databases. Nevertheless, a total grasp of the language of life requires a catalogue of the multidi- mensional information stored within eacln cell as the dynamic interactions between ions, metabolites, macromolecules and organelles. We have initiated the development of the Cellomics T M database as an ency- clopedic resource of cellular and molecular processes. Although combinatorial chemistry, ge:aomics and proteomics have become important elements in drug-target discovery, validation and homogeneous screening, we believe that cellular information, which includes all these elements, will be crucial to future drug-discover' paradigms. Fluorescent-protein biosen- sors have become an important tool in this cellular approach and are used as reagents to extract data from living cells with 'high-content' screens. These high-content screens deliver multiparameter data

K. A. Giuliano (giuliano@biodx.com) and D. L. Taylor are at Cellomics Inc. (formerly BioDx), 635 William Pitt H/ay, Pittsburgh, P,,t 15238, USA.

concerning the effects of lead compounds on specific molecular processes (Fig. 1).

P r o t e i n s as i d e a l b i o s e n s o r s Proteins have evolved to mediate the chemical reac-

tions within cells. Therefore, when converted to sen- sors of the intracellular milieu, proteins have the poten- tial to report not only the dynamic distribution of specific reactions but also data concerning reaction kinetics, protein interactions and post-translational modifications. One way to define the activity of pro- teins is through the environmental changes that occur either internally or on their surface, including binding to other proteins. Fluorescently labelled proteins designed to sense and report these changes have been termed 'fluorescent-protein biosensors'. These new reagents have evolved from the development of a variety offlu- orescently labelled macromolecules 1. The protein com- ponent of the biosensor provides a highly evolved mol- ecular-recognition moiety, and a fluorescent molecule attached to this protein component in the proximity of the active site transduces environmental changes into fluorescence signals that are detected using a system with an appropriate temporal and spatial resolution. Because the modulation of native protein activity within the living cell is reversible, and because fluor- escent-protein biosensors can be designed to sense reversible changes in protein activity, these biosensors are essentially reusable within the cell. The basic prin- ciples and initial applications of these fluorescent- protein biosensors have been described in detail else- where 1. Briefly, they can be designed to measure the conformational changes involved in the modulation of

Copyright © 1998, Elsevier Science Ltd. All rights reserved. C167 - 7799/98/$19.00. PII: S0167-7799(97)01166-9 TIBTECH MARCH 1998 (VOL 16)

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O O O 0 0 0 0 0 0 0 0 0 O 0 0 O O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 O Q O 0 0 0 0 0 0 0 0 0 O0000000C)O00 O0000000C)O00 0 0 0 0 0 0 0 0 0 0 0 0 OOO0000~)O00

Multiple-well / drug-screening / platform ~ D r u g - t r e a t e d

- f - - - - - - - - t -~ i well

Matrix interactions

Gene expression

Organellar dynamics

Enzyme localization / and activity

Translocations

Receptor-ligand interactions

°N ""-4

Signal transduction

Single-cell analysis

Figure 1 Fluorescent-protein biosensors and high-content screens in drug discovery. A high-content screen involving fluorescent-protein biosensors is shown here in schematic outline. A multiple-well drug;screening platform containing living cells is treated with a combination of drug types and concentrations. This platform can range from a standard 96-well plate with physical wells to a miniature transparent chip with 'virtual' wells; in either case, multiple livir g cells within each well are imaged over time both before and after drug treatment. The molecular processes within each cell are reported by fluorescent-protein biosensors and analysed in time and space. Shown at the single-cell level are examples of the processes that can be m~asured with fluorescent-protein biosensors. When combined in a single cell, several sensors can be used to measure and correlate the effects of lead compounds on multiple pathways.

enzymatic activity and the interaction of proteins with numerous other proteins, DNA, RNA, complex carbohydrates, lipids and organelles. These activity- dependent conformation:d changes fall into three gen- eral classes: (1) spatial rearrangements of the peptide backbone that alter the distance between specific amino acid residues; (2) activitT-dependent changes in the exposure of certain amino acid residues to solvent; (3) changes in the hydrodymmic radius of a protein (e.g. macromolecular shape changes or macromolecular complex formation). The distinctions between these general classes of protein interactions are not clear-cut and overlap between them is probably the rule rather than the exception. For instance, a change in the spa- tial distance between specific amino acid residues within a protein is likely to be accompanied by a change in protein shape or exposure of a previously hidden region of the protein to solvent, or both. Therefore, the signals derived from tluorescent-protein biosensors must be interpreted carefully, because these reagents can report a combination of environmental changes, especially in the complex living cytoplasm.

Just as protein-activity-dependent conformational changes can be classified in this general fashion, the fluorescent reagents and spectroscopic methodologies used to construct and detect fluorescent-protein

biosensors in living cells can also be loosely grouped. Three types of fluorescence spectroscopy can be used in conjunction with fluorescence-ratio-imaging microscopy, a major tool in quantifying fluorescence 1. The following ratio-imaging methods are, in addition, independent of cytoplasmic path length, accessible volume and local concentration. (1) The fluorescence- resonance-energy-transfer (FILET) and fluorescence- quenching techniques are sensitive to intra- and inter- molecular distances and can therefore sense relatively small changes in protein conformation and protein interactions. (2) Solvent-sensitive fluorescent reagents can detect intracellular changes in protein confor- mation and protein-ligand binding. (3) Fluorescence- anisotropy-imaging microscopy is sensitive to the rotational diffusion of labelled molecules 2.

Recent applications o f fluorescent-protein biosensors

Since we published our last reviews of fluorescent- protein biosensors 1,3, there have been many new applications of the approach. Table 1, which is not meant to be an exhaustive list of new applications, shows the range of biological activities that can be measured in vitro and in vivo using fluorescent-protein biosensors,

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Table 1. Recent applications of fluorescent-protein biosensors

Target Fluore.,;cence Sensor Environment Refs meas urement

Cell signalling: membrane processes Membrane potential FRET Fluorescent lectin and membrane-bound In vivo 4

oxonol dye Free Ca 2÷ near p l a s m a Luminescence Adenylyl-cyclase-aequorin chimera In vivo 5

membrane Insulin-receptor signalling FRET, a~isotropy Native and fluorescently labelled insulin In vitro 7 EGF-receptor signalling Solvent sensitivity Environmentally sensitive fluorescent In vitro 6

guanine nucleotides

Cell signalling: cytoplasmic processes Intracellular cAMP in neural FRET

circuits Protein kinase A Solvent sensitivity

Protein kinase C: translocation and fragmentation

Human glucocorticoid-receptor translocation

Calmodulin activation Intracellular free Ca 2÷

concentration Small-G-protein (cdc 42Hs)

signalling

Fluorescently labelled catalytic and regulatory subunits of protein kinase A

Environmentally sensitive fluorescent analogues of cAMP

Intensity redistribution Fluorescentlylabelled kinase-regulatory- domain plus antibody-labelled catalytic domain

Intensity redistribution Human-glucocorticoid-receptor-GFP In vivo 12,13 chimeras

FRET Bridged GFP-BFP chimera In vivo 15 FRET Bridged GFP-BFP chimeras In vivo 16,17

Solvent sensitivity Environmentally sensitive fluorescent In vitro 18 guanine nucleotides

FRET Energy transfer between fluorescently In vitro 19 labelled cdc42Hs and guanine nucleotides

Solvent :sensitivity Dansyl-amide interaction with carbonic- In vitro 22 anhydrase-II-bound Zn 2÷

Anisotro~)y Fluorescent sulfonamide interaction with In vitro 23 carbonic-anhydrase-II-bound Zn 2+

Solvent :~ensitivity Environmentally sensitive dyes coupled In vitro 24 to synthetic zinc-finger domains

FRET Energy transfer between two dyes In vitro 25 covalently bound to a zinc-binding peptide

Free Zn 2. concentration

Cell physiology Role of myosin-II phosphorylation

in cytokinesis and cell motility

In vivo 8

In vitro 9

In vitro and in vivo 10

Apoptosis-related proteolysis

Energy metabolism

FRET Energy transfer between two dyes In vivo 26 covalently bound to myosin-II heavy and light chains

Quenching Doubly labelled protease substrate In vivo 27 analogues

Two-photon excitation NAD(P)H fluorescence measurements in In vivo 29 pancreatic p-cells

FRET Energy transfer between NADH and In vivo 30 membrane-bound rhodamine 123

Abbreviations: BFP, blue fluorescent protein; EGF, epithelial growth factor; FRET, fluorescence-resonance energy transfer; GFP, green fluorescent protein.

Cell signalling Membrane processes

Some recently described fluorescent-p::otein bio- sensors have been designed to report chemical events occurring at or near the plasma membrane. Mem- brane-potential changes have been measured as a result of FRET between a labelled lectin and an oxonol dye whose dynamic partitioning in the plasma membrane was dependent on the electrical potential

across the membrane 4. In a live-cell chemilumines- cence application, a novel adenylyl-cyclase-aequorin chimera expressed within human kidney cells was found to be localized to the plasma membrane, where it reported free intracellular-Ca2+-concentration changes distinct from global changes within the same cell s. In vitro, the interaction of insulin and EGF with their respective receptors has been studied by combin- ing FRET, environment,'d sensitivity and anisotropy

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approaches with flumescently labelled receptor ligands 6,7.

Cytoplasmic processes Fluorescent-protein biosensors have become power-

fhl tools when applied to the processes involved in cyto- plasmic signal transduction. A wide range of cellular events involving ions, me~:abolites and macromolecules are being dissected with fl aorescent-protein biosensors. F1CRhl~, a protein-kinase-A-based reporter of the temporal and spatial distribution ofintracellular cAME has been used to show that the intracellular diffusion of cAMP may be involved in converting transient signals into long-term gene expression within the nervous system s . In vitro, the interaction between cAMP and protein kinase A has beets measured using fluorescent analogues of cAMW. The intracellular translocation and fragmentation of another signal-transduction kinase, protein kinase C 13I, has been measured using a fluorescent analogue of the enzyme and domain- specific antibodies ~°.

In our last review" of the field, we proposed that the use of molecular biology to incorporate fluorophores into proteins to produce biosensors would be comple- mentary to biosensors produced in vitro before intro- ducing them into living zellsL Since then, dozens of papers describing the intracellular localization of green- and blue-fluorescent-protein (GFP and BFP) chimeras have appeared; the use of GFP in cell biology and biotechnology has recentiy been reviewed 11. Some of the fluorescent-protein chimeras have been used to map the dynamic redistribution of receptor-ligand complexes12 14. BFP-GFP dual chimeras have also been designed as fluorescent-protein biosensors to report on intracellular protein activation. A FRET-based sensor ofcalmodulin activation was formed by juxtaposing the calmodulin-binding pep:ide sequence of smooth- muscle myosin-light-chain kinase between BFP and GFP molecules is. Binding ofa Ca2+-calmodulin com- plex to the sensor altered its FRET spectrum, a change measurable with fluorescence-ratio imaging 1. The BFP-GFP-based fluorescent-protein biosensor is much like the calmodulin-activation sensors previously reported 1, in that it was produced in vitro and microin- jet ted into living cells, in which the analysis took place. FRET-based sensors of free intracellular Ca 2+ concen- tration have been prepared in a similar manner by inserting the Ca2+-bindi:ng site of calmodulin itself between the taro protein-based fluorophores >'17.

To understand the role that the small G-proteins play in signal transduction, two cdc42Hs sensors have been designed for in vitro use. One sensor used environmen- tally sensitive, fluorescent guanine-nucleotide ana- logues to measure the interaction of cdc42Hs with other proteins TM. The other, designed to measure gua- nine-nucleotide-exchange kinetics, was based on FRET between a fluorescently labelled cdc42Hs mol- ecule and a fluorescent analogue of a guanine nucleotide 19.

The above examples show that several fluorescence- based approaches can be used to sense the activity of

a protein molecule, but none demonstrates the ver- satiety of the molecular fluorescence approach better than protein-based zinc biosensors. Small-organic- molecule-based fluorescent sensors offree-intracellular- zinc concentration have been known for years 2°,21, but concerns over avidity, specificity and intracellular loc- alization have prevented their widespread use. More recently, there have been no-less-than-four distinct fluorescence approaches used to sense physiologically relevant free-zinc concentrations.

Two of these were based on the interaction of the metal ion with carbonic anhydrase II, a zinc-binding enzyme; one of these used fluorescence intensity and wavelength measurements to detect the interaction of dansyl-amide molecules with carbonic-anhydrase-II- bound zinc 22, and the other used fluorescence- anisotropy to measure the interaction of a fluorescent sulfonanfide with carbonic-anhydrase-II-bound zinc ions 23. The other two were based on peptides that bind zinc ions with great avidity and undergo a precise con- formational change upon metal-ion binding; one used enviromnentally sensitive fluorescent dyes to report the conformational changes 24, while the other used FRET with a doubly, labelled peptide to sense zinc binding 2s.

Cell physiology This loosely grouped category contains applications

of fluorescent sensors to the measurement of intra- cellular contractility; proteolytic activity and energy metabolism. In one example, a fluorescent analogue of smooth-muscle myosin II and a fluorescent-protein biosensor of myosin-II-light-chain phosphorylation were used to dissect the role that myosin II plays during cytokinesis and cell motility. The system tested models of cell division and locomotion that could not have been tested within living cells before the development of macromolecule-based fluorescent reagents 26. In another example, intracellular protease activity, was measured with a fluorescently labelled peptide substrate 27. An intact: hydrophobic U-shaped peptide maintained two fluorophores in a stacked conformation that quenched their fluorescence. Once a specific protease cleaved the peptide into two independent chains, they diflfiased away front each other, thus permitting the dye attached to each chain to become fluorescent. The temporal activation ofproteases during cellular processes such as apoptosis was therefore measured as an increase in fluorescence intensity ~ of the entire cell. This approach is similar to that described for 'molecular beacons', fluorescent nucleic acid probes whose hybridization to a comple- mentaw sequence relieves the quenching of the fluor- escent probe as it is converted from a hairpin structure to a linear conformation :s. Finally, energy metabohsm in living cells has been measured using the fluorescent form of the metabolic electron-shuttling molecule NAD(P)H. In one set o f experiments, two-photon excitation of fluorescence was used to measure NAD(P)H concentration in specific planes within liv- ing cells29; in another, FRET between NAD(P)H and membrane-bound rhodamine 123 was used to test the

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effect of oxidative-phosphorylation ink.ibitors on mitochondrial metabolism 30.

Improvements in site-specific fluorescence lebelling of proteins

The design of successful fluorescent-protein biosen- sors depends strongly on fluorescently labelling the tar- get protein at specific sites ~, and a number of strategies to accomplish this task have been descril:ed. In one approach, unique cysteine residues have been intro- duced into regions of mutated maltose-binding pro- reins that are known to be allosterically linked to the active site 31. When the sulfhydryl groups ofl:he inserted cysteine residues were modified with environmentally sensitive fluorescent dyes, a series of fluorescent- protein biosensors were produced that were capable of measuring substrate concentrations in vitro over a range of five orders of magnitude. In a similar manner, P-glycoprotein was specifically labelled at two conserved cysteine residues to produce a fluorescent-F.rotein bio- sensor to measure the conformational communication between the drug-binding site(s) and ATPase catalytic site on the protein 32. Moreover, two conserved cysteine residues in cardiac troponin C have be,m labelled with environmentally sensitive dyes to produce a fluorescent-protein biosensor of the molecular events involved in muscle contraction. ExperimerLts with the sensors were performed in vitro and in skinned cardiac muscle fibres 33.

Two molecular techniques of site-specific protein labelling with fluorescent probes have recently been described. In the first, 'unnatural' or nonsense amino acid mutagenesis was used to introduce a ketone- containing amino acid into a protein during in vitro translation 34. The synthetic protein was then labelled specifically at the ketone amino acid residue using a fluorescent hydrazine dye. In a second approach involv- ing in vitro protein translation, a fluorescent amino acid analogue was inserted directly into a specific site of newly synthesized [3-galactosidase 3s.

Fluorescent-protein biosensors should therefore con- tinue to have an important impact on d~fining the chemical and molecular dynamics responsible for cell and tissue functions. The recent explosion i:a the appli- cation of this approach is due mainly to the use of GFP to trace protein distribution and activity in living cells. The simplicity of protein-chimera preparation is largely responsible for the success of GFP as a sensor. Further development of new classes of extrinsic luminescent probes and the continued evolution of powerful instru- mentation to measure temporal and spatial dynamics in living cells will be combined at a molecular level with GFP-based sensors to dissect life processes that simply cannot be reconstituted in vitro.

Fluorescent-protein biosensors and the future o f drug discovery

A recent review of biosensing technology and its application to the pharmaceutical industry reported examples of fluorescence-based biosensors designed for in vitro analyte measurement 36. Furthermore, it

suggested that many biosensors have found application in quantifying a drug substance in the finished drug product 36. We believe that fluorescent-protein biosen- sors and their live-cell applications will be useful much earlier in the drug-discovery process. When applied to target validation and lead optimization, fluorescent- protein biosensors will become essential reagents in strategies for breaking the bottlenecks in the drug- development pipeline. We have recently described a high-content screen that coupled a GFP-human- glucocorticoid-receptor chimera with the ArrayScan TM

plate-reader system to measure the translocation of ligand-receptor complexes from the cytoplasm to the nucleus in drug-treated cells 37.

As drug targets, fluorescent-protein biosensors have the great advantage that lead-compound screening automatically occurs within the environment in which the final product has its pharmacological effect, the liv- ing cell. When fluorescent-protein biosensors are com- bined with other live-cell-based fluorescent reagents and applied to target-validation and lead-optimization strategies, they compose what we have termed high- content screening (HCS) 37. Briefly, HCS consists of multiparametric measurements of the effects that lead compounds have on the temporal and spatial distribu- tion and activity of intracellular ions, metabolites, macromolecules and organelles. Thus, the entry of a lead compound into the cell, its effect on a specific tar- get and other cellular constituents in time and space, and its toxicity can all be simultaneously measured and correlated using HCS.

The evolution of fluorescent-protein biosensors within the context of drug discovery promises exciting possibilities. We are now witnessing the engineering of endogenous cellular components that constitutively localize and function as reporters of their own activity. Single cells have been used as biosensors for chemical separations 3s. Continuous cell lines that produce endogenous fluorescent-protein biosensors can them- selves be envisioned as living reagents. As huge com- binatorial libraries of potential lead compounds begin to produce thousands of high-throughput screening 'hits', fluorescent-protein biosensors will become key components of HCS strategies to ease bottlenecks in the evolving drug-discovery process.

Note added in proof A new GFP biosensor has recendy been reported. A

fluorescent-protein chimera containing [3 arrestin has been used to detect G-protein-coupled-receptor activ- ation in living cells. (Barak, L. S. et al. (1997)J. Biol. Ckem. 272, 27497-27500.)

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