Recent Advances of Fluorescent Technologies for Drug Discovery and ...

Current Pharmaceutical Design, 2009, 15, 000-000

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Recent Advances of Fluorescent Technologies for Drug Discovery and Development Chiranjib Chakraborty*, Chi-Hsin Hsu, Zhi-Hong Wen and Chan-Shing Lin1 Department of Marine Biotechnology and Resources, College of Marine Science and Division of Marine Biotechnology, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohisung, Taiwan Abstract: Recent progresses in the development of fluorescent technologies become a reliable device for drug discovery research. The fluorescence tools offer attractive options for an opportunity to visualize the effects of drug candidates in the cells. The fluorescent tools, such as fluorescent protein, are regularly used in a range of drug discovery processes. A better understanding and use of fluorescent technologies facilitate drug discovery research faster and can open up new applications. Therefore, we have provided information about some new generation fluorescent reagents (GFP and fluorophores). This review illustrates how fluorescent technologies and fluorescent tools are contributing to the drug discovery process mainly high-throughput screening (HTS), disease mechanism based target discovery, disease-genes-based target discovery, ‘target classes’ based target candidate discovery, physiology-based drug discovery, genomics-based drug discovery, target validation and their future perspectives.

Keywords: Fluorescent technologies, drug discovery, novel classes of drugs, progress in drug development INTRODUCTION Many research tools have been developed and popularized over the last decade. Among them, important one is the fluorescent tools and techniques (Fig. 2). Several new techniques, probes and equipment have been developed for fluorescent research; as a result, the evolution of this field is very rapid. A quick word search ‘fluorescence’ in PubMed for example yield 127,804 articles in 2004 [1] and in 2006, the number jumped up to 187,936. At the end of the 2008, the number was 222,841 (Fig. 3). This indicates how researchers are adapting to ‘fluorescent research tool’ rapidly. Fluorescence-based techniques are crucial to study cellular mechanism as well as cellular structure and function, and interactions of molecules in biological systems for drug discovery (Fig. 1). Presently, fluorescence is essential in the detection and quantification of nucleic acids and proteins in gel electrophoresis, microarrays, and fluorescence spectroscopy. During the last few years, several new companies and chemists have started to develop large number of fluorescent particles or probes that are applicable for labeling, localized any probable aspect of biological system. A good example is Invitrogen that can provide 3,000 or more fluorescent probes with their application information. With the availability of a large range of fluorophores, researchers have started to apply these fluorescent probes in cellular, sub-cellular research and many other different fields. Furthermore, the fundamentally fluorescent gene products, most popular green fluorescent protein (GFP) and its variants, have accepted molecular biologists to visualize the tag protein components into living systems and lead in a new era for fluorescence.

*Address correspondence to this author at the Department of Marine Biotechnology and Resources, College of Marine Science and Division of Marine Biotechnology, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohisung, Taiwan; E-mail: [email protected]; [email protected]

1381-6128/09 $55.00+.00

During the last decade, fluorescent techniques and technologies become a reliable tool for pharmacological research as well [2-3] (Table 1-3) (Fig. 4). They provide novel insights into compound-related responses in drug discovery. From drug candidate screening to clinical trial, fluorescent molecules are essential tools to contribute novel therapeutic compounds. Researchers are beginning to utilize wide range of fluorescent proteins or probe to study diverse biological processes within cells to discover new targets. It has been used to find out the receptor site localization and expression using fluorescent proteins or probe to discover novel potential drug candidates. In this review, we have presented different fluorescent probes and technologies, and describe how they can be helpful in the drug discovery practice. We have also discussed how fluorescent methods has contributed to drug discovery and development especially in high throughput screening, discovery of new targets, validation of targets and the future prospective of fluorescent research in drug discovery. HIGH THROUGHPUT SCREENING Presently high-throughput screening (HTS) is a frequently used method for drug discovery, especially to quickly evaluate the activity of a large number of compounds or extracts on a target. Purpose Millions of biochemical, genetic or pharmacological tests can be conducted very quickly through HTS. Through this process one can rapidly identify active compounds, antibodies or genes which modulate a particular biomolecular pathway. The results of these experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology.

© 2009 Bentham Science Publishers Ltd.

2 Current Pharmaceutical Design, 2009, Vol. 15, No. 00

Fig. (1). Flow chart of drug discovery and development process.

Chakraborty et al.

Recent Advances of Fluorescent Technologies for Drug Discovery

Current Pharmaceutical Design, 2009, Vol. 15, No. 00

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Fig. (2). Fluorescence fundamentals. (A) At the very beginning, energy is absorbed by the atom which becomes excited. Then, the electron jumps to a higher energy level. Soon, the electron drops back to the ground state, emitting a photon (or a packet of light) - the atom is fluorescing. (B) There are three stages for fluorescence is the emission. First,absorption and excitation where a photon of energy hv EX is supplied by an external source such as by a lamp or a laser and absorbed by the fluorophore, creating an excited electronic singlet state (S1'). This process distinguishes fluorescence from chemiluminescence, in which the excited state is populated by a chemical reaction. Second, excited-state where The excited state exists for a finite time (typically 1-10 nanoseconds).During this time, the fluorophore undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. Third, fluorescence emission where A photon of energy h EM is emitted, returning the fluorophore to its ground state S0. Due to energy dissipation during the excited-state, the energy of this photon is lower, and therefore of longer wavelength, than the excitation photon h EX. The difference in energy or wavelength represented by (h EX - h EM) is called the Stokes shift.

Fig. (3). Increase in the use of fluorescence research reported in Pubmed references from the year 2004 to 2008. The term ‘fluorescence’ was used for searching in Pubmed.


Chakraborty et al.

Fig. (4). Schematic diagram of different fluorescent technologies, which have been used regularly for drug discovery and development processes.


Table 1.


Different Fluorescence Instruments Used for Drug Discovery Research Uses

Fluorescence Instruments Fluorescence microscopes (FM):

Spectrofluorometer :

Microplate reader:

•

It is a light microscope and based on the occurrence that certain material emits energy noticeable as visible light when irradiated with the light of a specific wavelength. The sample can either be fluorescing in its natural form like chlorophyll and or treated with fluorescing chemicals.

•

The specimen for FM is illuminated with light of a specific wavelength(s) which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Typical components of a fluorescence microscope are the light source (xenon arc lamp or mercury-vapor lamp), the excitation filter, the dichroic mirror (or dichromatic beamsplitter), and the emission filter. The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen.

•

The spectrofluorometer is an instrument which takes advantage of fluorescent properties of some compounds in order to provide information regarding their concentration and chemical environment in a sample. A certain excitation wavelength is selected, and the emission is observed either at a single wavelength or a scan is performed to record the intensity versus wavelength also called an emission spectra.

•

This instrument can evaluate the standard properties of bulk samples.

•

Microplate Readers are designed to detect biological, chemical or physical events of samples in microtiter plates. This instrument can evaluate the standard properties of bulk samples. Sample reactions can be (assayed) in 6-1536 well format microtiter plates. In most cases, a high-intensity lamp passes light to the microtiter well and the light emitted by the reaction happening in the microplate well is quantified by a detector. Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization

•

Microplate Detection may used for: ELISA; protein and cell growth assays; nucleic acid quantitation; molecular interactions; enzyme activity; cell toxicity, proliferation, and viability; atp quantification; immunoassays; high throughput screening of compounds and targets in drug discovery such as flipr assays

Fluorescence scanners and Microarray readers/ scanners:

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These instruments can evaluate resolution of fluorescence in two dimensions macroscopic objects like electrophoresis gels, blots and chromatograms including microarray.

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Most microarray manufacturers, such as Affymetrix and Agilent, provide commercial data analysis software with microarray equipment such as plate readers with scanner which scans fluorescence specimens.

Flow cytometers:

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It can identify, count, examine and evaluate fluorescence per cell in a flowing stream, allowing subpopulations within a large sample.

Table 2.

Commercially Available Fluorescent Instruments, Software, Proteins and Probes Company

Available fluorescent instruments /software

Amaxa

Available fluorescent proteins

(www.amaxa.com)

pmaxFP-(Green); pmaxFP-(Yellow) pmaxFP-(Red)

Clontech

AmCyan(Cyan) AcGFP1(Green)

(www.clontech.com)

ZsGreen1(Green) ZsYellow1 (yellow)

Edinburgh Instruments (www.edinst.com)

spectrofluorometer, Fluorescence Analysis Software, Fluorescence microplate reader

Evrogen

PS-CFP2(Cyan), TurboGFP(Green), phiYFP(Yellow), JRed

(www.evrogen.com)

(Red) IBH - Fluorescence (www.ibh.co.uk)

Fluorescence Spectroscopy, Spectrofluorometers (FluoroMax®-4, FluoroLog®-3) Fluorescence based Forensic & Identification Systems

Available fluorescent probes/fluorophores

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Chakraborty et al.

(Table 2) Contd….

Company

Available fluorescent instruments /software

Invitrogen (www.invitrogen.com) ISS Inc (www.iss.com/)

HORIBA Jobin Yvon (www.JobinYvon.com)

Available fluorescent proteins

Available fluorescent probes/fluorophores

BFP(blue), CFP (Cyan), YFP(Yellow), GFP(Green)

Fluorescein, Alexa Fluor 405 Dye, Conjugates of the pyrene succinimidyl esters (P130, P6114, P6115) etc.

Dual channel spectrometer like Alba FCS™ , computer-controlled instrument specifically designed for confocal imaging applications using either single- or multi-photon excitation like Alba FLIM™ Spectrofluorometer, Fluorescence Detectors, Fluorescence software etc.

Lux Biotechnology

DsRed-Express (Red)

(www.luxbiotech.com)

DsRed-Monomer (Red)

Marker Gene Technoligs

Trifluoromethylumbelliferone (TFMU), Resorufin, Fluorescein, Rhodol , Rhodamine 123 , Propidium Iodide , 5(Octadecanoylamino)fluorescein etc.

(www.markergene.com)

MBL International

Midoriishi-Cyan

(www.mblintl.com)

Azami Green Kusabira-Orange

NanoLight Technology

RmGFP(Green)

(www.nanolight.com)

PtGFP(Green) RrGFP(Green)

Nikon

Microscopes and imaging devices

(www.nikon-instruments.com) Olympus


(www.olympusmicro.com) Perkin Elmer Life Sciences (www.las.perkinelmer.com)

Fluorescence Spectroscopy, microarrays reader, fluorescence microplate reader

Promega

RrGFP(Monster Green)

(www.promega.com) PubSpectra (www.home.earthlink.net/~pubspec tra)

Fluorescent Spectra Graphing Site; fluorophores information software

Stratagene

hrGFP(Green)

(www.stratagene.com) TauTheta Instruments (www.tautheta.com/)

Multifrequency phase fluorometers (MFPF) ;fluorescence sensors

Turner BioSystems (www.turnerbiosystems.com)

Spectrofluorometers and microplate readers

Zeiss


(http://www.zeiss.com)


Table 3.


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Fluorescence Techniques Commonly Used for in Drug Discovery

Technique/assay/Method

Applications

Therapeutic area

Green fluorescent protein microplate assay (GFPMA)

High throughput screening of compounds against Mycobacterium tuberculosis

Tuberculosis or any other anti bacterial drug discovery

Luminescence resonance energy transfer (LRET)

High-throughput screening (HTS) for crude naturalproduct library

Anti bacterial drug of infectious bacteria

Low-magnification fluorescence microscopy (Resolution: 500 m- 1mm)

Image and Entire cell populations: phenotype, proliferation, toxicity, and angiogenesis

Oncology and cardiovascular disease

Medium-magnification fluorescence microscopy (Resolution: 10-50 m)

Subpopulation analysis: individual cell phenol type like cell out growth

Neurology, metabolic diseases and cardiovascular disease

High-magnification fluorescence microscopy (Resolution: 1 m)

Intracellular molecular events: nuclear translocation, micronucleus formation

Oncology, inflammation and neurology

Fluorescence resonance energy transfer (FRET)

Based compound screening and target identification

For any diseases, like for cancer -proteases such as trypsin and caspase-3 assy

Bioluminescence resonance energy transfer (BRET)

Target identification

Several clinically significant disease

Flow cytometry (Resolution: 1-30 m )

Subpopulation image analysis or intracellular modifications: surface marker regulation, whole blood cells, phenotypic changes and proliferation

Autoimmunity, inflammation and cardiovascular disease

Homogeneous, time-resolved fluorescence (HTRF)

High throughput screening

High-throughput screening

Time-resolved fluorescence (TRF) and homogeneous TRF

Screening of chemical compounds

Anticancer drug screening

Reflection fluorescence microscopy (TIRFM)

Imaging for target identification


Voltage/ion probe reader (VIPR)

Monitor real-time target activity and screen large chemical libraries


Fluorescent membrane potential probe kit

Screen molecules potassium channel closers (KCCs) and openers (KCOs) in insulin- and glucagon-secreting cell lines

Type-I and Type-II Diabetes

Plate-based assay with fluorescent microarray

Ultra high-throughput screening (UHTS) of compounds


Fluorogenic PCR

To detect the gene mutation

Genetically inherited diseases

Method Using robotics, data processing software and sensitive detectors, HTS allows a researcher to scan the activities from whole genomics in a reasonable time and efforts. When assays are running in a parallel approach using multi-well assay plates, the term HTS is then used. Microtiter plate is a small container, usually disposable and made of plastic, that contain grid of small, open wells. Modern microplates for HTS generally have either 384, 1536, or 3456 wells that are all multiples of 96, but in the dimension of the original 96 well microplate,8 x 12 mm spaced wells. Most of the wells contain experimentally useful matter, often potentially bioactive compounds dissolved in an aqueous solution of dimethyl sulfoxide (DMSO), of which thousands of compounds different for each well across the plate can be tested at once. Depending on the results of this first assay,

the researcher can perform follow up assays within a small set of likely positive from the source wells that gave interesting results (known as "hits") into new assay plates, and then re-running the experiment to collect further data on this narrowed set, confirming and refining observations [4,131,132]. Unique distributions of compounds across one or many plates can be employed to increase either the number of assays per plate, or to reduce the variance of assay results, or both. The simplifying assumption made in this approach is that any N compounds in the same well will not typically interact with each other, or the assay target, in a manner that fundamentally changes the ability of the assay to detect true hits. For example, a plate where compound A is in wells 1-23, compound B is in wells 2-3-4, and compound C is in wells 3-4-5. In an assay of these plates against a given target, a hit


Table 4.

Chakraborty et al.

Fluorescence Techniques Commonly Used for in Drug Discovery

Technique/assay/Method

Strategies

Instruments Required

Green fluorescent protein microplate assay (GFPMA)

GFPMA used for high-throughput screening and kinetic monitoring

Microplate with a multiplate reader; microplate fluorometer

Luminescence resonance energy transfer (LRET)

LRET used for high-throughput screening

96-well plate with a multiplate reader

fluorescence microscopy

fluorescence microscopy

(Low-magnification, Mediummagnification,

to visualize the images

Fluorescence microscopy different lens resolution. Low-magnification (Resolution: 500 m- 1mm) Mediummagnification

High-magnification)

(Resolution: 10-50 m) High-magnification (Resolution: 1 m) Fluorescence resonance energy transfer (FRET)

FRET is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon

fluorescence microscopy, fluorescence confocal laser scanning microscopy

Bioluminescence resonance energy transfer (BRET)

BRET uses a bioluminescent luciferase that is genetically fused to one candidate protein, and a green fluorescent protein mutant fused to another protein of interest. Interactions between the two fusion proteins can bring the luciferase and green fluorescent protein close enough for resonance energy transfer to occur, thus changing the color of the bioluminescent emission.

Microcentrifuge, fluorescence microscopy,

Flow cytometry

Flow cytometry uses the principles of light scattering, light excitation, and emission of fluorochrome molecules to generate specific multiparameter data from particles and cells in the size range of 0.5um to 40um diameter

Flow cytometers

Time-resolved fluorescence (TRF)

Time-resolved fluorometry (TRF) takes advantage of the unique properties of the rare earth elements called lanthanides. The commonly used lanthanides in TRF assays are samarium (Sm), europium (Eu), terbium (Tb), and dysprosium (Dy). Because of their specific photophysical and spectral properties, complexes of rare earth ions are of major interest for fluorescence applications in biology.

Fluorescence microplate reader

Homogeneous, time-resolved fluorescence (HTRF)

HTRF uses the principles of both TRF and FRET. The HTRF donor fluorophore is either Europium cryptate (Eu3+ cryptate) or Lumi4™-Tb (Tb2+ cryptate).

fluorescence microscopy, fluorescence confocal laser scanning microscopy or fluorescence microplate reader

Total internal reflection fluorescence microscope (TIRFM)

TIRFM is a type of microscope with which a thin region of a specimen, usually less than 200 nm, can be observed.

Total internal reflection fluorescence microscope

Voltage/ion probe reader (VIPR)

VIPR instrumentation that are used to monitor real-time target activity and screen large chemical libraries

Microplate, fluorescence microplate reader

Fluorescent membrane potential probe kit

commercially available fluorescent membrane potential probe kit

Microplate, fluorescence microplate reader

fluorescent microarray

Microarray technology is a powerful tool to investigate global changes in gene/protein expression of cells and tissues using fluorescent dyes

microarray slide, Microarray cover slips, centrifuge, UV-vis Spectrophotometer, Water bath

Fluorogenic PCR

fluorescent dye labeling of PCR fragments

PCR

(Resolution: 1-30 m)

in wells 2, 3, and 4 would indicate that compound B is the most likely agent, while also providing three measurements of compound B's efficacy against the specified target. Commercial applications of this approach involve combi-

nations in which no two compounds ever share more than one well, to reduce the (second-order) possibility of interference between pairs of compounds being screened.


Applications in Drug Discovery The development of new antimicrobial drugs is a scientific challenge because of increasing number of antibiotic-resistant pathogenic microorganisms. A green fluorescent protein microplate assay (GFPMA) for the high throughput screening of compounds against Mycobacterium tuberculosis has been developed [4] .The plasmid pFPV2 containing the gene for EGFP, has been transformed into two strains of M. tuberculosis H37Ra and H37Rv by electroporation. The minimum inhibitory concentration (MIC) of various anti-mycobacterium agents have been determined using this GFPMA and have been compared with those obtained using the conventional Mycobacterium testing BACTEC 460 radiorespirometric assay system. Same type of another GFPMA assay has been performed for the high throughput screening of compounds against Mycobacterium tuberculosis H37Rv, H37Ra, and Erdman strains with the pFPCA1, which contains a red-shifted gfp gene [5]. The employment of GFP marker protein provides a rapid, useful platform for routine high-throughput screening of antituberculosis compounds. Luminescence, which emits light without heat, occurs at low temperature. The lumination can be caused by chemical, biochemical, or crystallographic changes, electrical energy, subatomic motions, reactions in crystals, and stimulation of an atomic system. Fluorescence and phosphorescence are two types of luminescence which are distinguished by the delay in reaction to external electromagnetic radiation. The phenomenon observed in ancient time like phosphorescence in the form of a glow emitted by the oceans at night, and this phenomenon was confused with the burning of the chemical phosphor, but, in fact, phosphorescence has nothing at all to do with burning. Likewise, fluorescence, as applied today in fluorescent lighting, involves no heat—thus creating a form of lighting more efficient than that that comes from incandescent bulbs. Several kinds of luminescence are known: chemoluminescence, bioluminescence, crystalloluminescence, electroluminescence (cathodoluminescence), photoluminescence (phosphorescence and fluorescence), radioluminescence, sonoluminescence, thermoluminescence, triboluminescence. Luminescence resonance energy transfer (LRET) is a type of luminescence phenomena in which excited energy is transferred from one source to the second substance; this assay is used regularly for high-throughput screening (HTS) in drug discovery. An assay for Escherichia coli sigma subunit binding to RNA polymerase based on luminescence resonance energy transfer (LRET) has been developed by using a europium-labeled 70 and an IC5labeled fragment of the ’ subunit of RNA polymerase. The LRET has been effectively used to screen a crude naturalproduct library. The LRET with technical advances offers a very robust assay suitable for high-throughput screening; this method is a potent tool for the investigation of any essential protein-protein interaction and drug discovery [6]. Enhanced green fluorescent protein (EGFP) is regularly used in HTS to compound screening. An assay using EGFP has been developed to screen new platinum-based antitumor drugs where EGFP has been used as a reporter. Platinum compounds such as cisplatin and carboplatin have been regularly used for chemotherapy to the cancer patient, but


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many tumors are resistant to these compounds. To address this issue, one assay named ‘Tet-On system’ has been used where the gene encoding EGFP has been cloned into a plasmid with a human cytomegalovirus (CMV) promoter. With the adding of doxycycline, the tet-responsive transcriptional activator binds and activates the tetracyclineresponsive element, which then activates the CMV promoter. Furthermore, EGFP expression has been induced by the addition of doxycycline to the medium. The plasmid containing EGFP with CMV promoter has been transformed into human cancer cells. A dose-dependence decreases in EGFP expression. For the application, an assay has been developed with trans platinum compounds that stimulate stress on cells and can lead to boost of the GFP expression. Cis platinum compounds form 1,2-intrastrand cross-link and cause differential recognition of the proteins involved in transcription that leads the inhibition of the EGFP expression [7]. Further, this fluorescent method helps us to screen antitumor drugs that can be completed within 24 h in a high throughput plan. Infection by HIV-1 virus can be monitored by an anti-p24 (core protein) in ELISA plates, which is a time consuming and expensive method. Using EGFP as a reporter, Gervaix et al. [8] has developed an alternative assay, where a similar increase in p24 antigen level has been observed by ELISA, which indicates that HIV-1 replication is likely represented by LTR-control expression of EGFP. Fluorescence microscopy, cytofluorimetry, and flow cytometry have been used to monitor fluorescence intensity of cells; therefore, the EGFP-based assay is very simple and direct method to detect HIV-1 virus in the cells.. Drug susceptibility has been determined by adding several molecules like inhibitors of reverse transcriptase, protease, as well as other targets. This assay has potential applications in HTS of anti-HIV drugs as it is fast, easy and economical, and fluorescence cell sorting is also possible with this method without adding of external substrates or labels. One of the limitations in using GFP as a transcriptional reporter is its half-life which is longer than 24 h. High halflife of reporter can obstruct the accurate determination of the induction level due to high background, especially in the assays where changes in the mRNA level are reflected by the changes in the reporter protein expression. This drawback has been prevailed over by constructing a mutant called as destabilized EGFP (d2EGFP), which last 2 h of half-life. Fusion vectors have been developed between the nuclear factor -binding protein with either d2EGFP or EGFP. An enhance level in fluorescence has observed from the pNFBd2EGFP containing cells in compare to those containing pNF-B-EGFP, after addition of the activator tumor necrosis factor [9]. Fluorescence resonance energy transfer (FRET), a distance-dependent interaction between the electronic excited states of two dye molecules, is used for HTS-based compound screening. Several FRET-based assays have been developed using GFP or mutants of GFP for proteases, such as trypsin and caspase-3, and for Ca2+ determination, as well as for the monitoring of protein-protein interactions [1014]. Moreover, the increasing availability of GFP mutants fulfills the increased demand for FRET assays and provides the homogeneous nature of the assay. Several other assays have been reported using GFP and other fluorescent


molecules that can potentially be applied to homogeneous or heterogeneous HTS assays [3, 15]. Homogeneous assay method is a method in which all the components of the assay are present during measurement. The reactions occur in solution generally without a solidphase attachment. In heterogeneous assay, some components in the assay are added or removed sequentially during the process, so that the measurements are made from a solid surface. Both assays are frequently used in drug discovery and development process. Homogeneous assays, which keep away from steps like coating and washing the plates, offer several advantages over solid-phase type assays, like- components can be pipetted, incubated and measured directly in the microplate- reduced time and cost. A range of homogeneous assay technologies are accessible using a broad range of targets such as receptors, enzymes, immuno-assays, and cellular assays. All these are developed by using GFP and fluorescent molecules; for example, binding assays, FRET-based assays, and cellular assays [16, 17]. A homogeneous assay for -lactamase has been developed using a protein engineering approach by designing a GFP fused with a molecular recognition site for -lactamase-inhibitory protein (BLIP) [18]. A new format of homogeneous assay has also been developed called homogeneous, time-resolved fluorescence (HTRF). A pair of fluorescent compounds to label biomolecules can be used for assay system. This technique combines the advantages of homogeneous assay formats, the robustness and sensitivity of radioisotopic methods, and the safety and stability of non-isotopic fluorescent labels. It can be useful in a high throughput screening format [19, 20]. Presently, growing interest in kinases as potential targets for therapeutic intervention has prompted the development of many kinase assay technologies. So far, for in vitro kinase assays, the applications of this technology are mainly paying attention on HTS [21, 22]. Abbott Bioresearch Center has extended the applications of HTRF technology to the areas of enzyme and inhibitor characterization [23]. Previously, an automated instrument has been developed by Abbott Laboratories called ARCHITECT i200 that can perform 200 chemiluminescence-based immunoassays per hour with up to 25 reagents on board. These 96- or 384-well based solid phase assays can be automated by this method. Hence, heterogeneous assays also play a key role in HTS. One of the important factors in these types of assays is the sensitive label. In this point of view, a number of heterogeneous assays are being developed using GFP and fluorescent molecules. For the detection of two peptidic analytes, an assay has been developed using mutants of GFP [24]. An assay to measure a peptide secreted by transfected CHO cell monolayers has been also designed as a sandwich ELISA utilizing streptavidin labeled horseradish peroxidase (HRP). To enhance the signal and the stability of the reagents, HRP has been changed to Eu+3 based DELFIA (dissociation enhanced lanthanide fluoroimmuno assay) with timeresolved fluorescence (TRF). Though, this set-up gives a good signal-to-noise; a one-step homogeneous TRF (HTRF®) format has been adopted to reduce the assay time and steps. The signal is comparable between TRF and HTRF®. The HTRF® assay has been demonstrated in screening leading drug in 384-well plates [25].

Chakraborty et al.

Assays run in 1536-well plates with tiny volumes (singledigit microliter to nanoliter scale) are referred to ultra highthroughput screening (UHTS). One of such type UHTS technology is developed by the Reaction Biology Corporation (USA), which is micro fluidic and offers highly productive and low-cost screening with high throughput selectivity profiling. This plate-based assay is less expensive to operate with a unique microarray system that runs over 6,000 homogeneous reactions per 1" x 3" microarray using very little amount, such as 1-nl volume, of compound dilutions and chemical libraries. The fluorescent signals can be detected easily and analyzed with conventional microarray scanners and software [26]. Another UHTS is aiming to use in pharmacological screening for plant drug discovery, competitive with combinatorial chemistry where transgenic plant cells are used for "in situ screening." A mammalian receptor protein, linked to a reporter construct, such as green fluorescent protein, is expressed and used for that system. Clonal cultures that produce ligands for this receptor can now be rapidly recognized through an UHTPS [27]. BODIPY, an erythromycin probe of bacterial ribosomes, has been designed and synthesized by Cumbre Inc. (USA). By attaching a BODIPY fluorophore to the 4'’- and 9’-positions of the erythromycin structure, the synthetic fluorescent probes have been effectively adapted inUHTS to identify several novel ribosome inhibitors [28]. Another UHTS has been developed by Interactions and New Assay Technologies, (a division of SmithKline Beecham Pharmaceuticals, UK), which is homogeneous fluorescencebased binding assay and highly amenable to miniaturization [29]. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method, which is susceptible to brightness changes of each particle, like those induced by binding fluorescent ligands to membrane particles with multiple receptor sites. This FIDA method has been performed along with fluorescence anisotropy (FA) and FA is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. DISEASE MECHANISM DISCOVERY

BASED

TARGET

From the disease mechanism, we can understand the possible causes and pathway of a particular disease. Understanding the disease mechanism can help us for possible treatment to slow or repeal the disease process. Disease mechanism can be broadly classified into the five groups which are defects in distinct genes or genetic disorders; infection by microorganisms like bacteria, fungi, or viruses; immune or autoimmune disease; trauma and acute disease based on injury or organ failure; multi-causal disease. Recently, a review has proposed mechanism-based target identification and drug discovery for cancer, a multi-causal disease. Research is in progress for mechanism-based target identification where fluorescent technology is involved [30,31]. Huntington's disease (HD) is an example of genetic disorders class of diseases. It’s an inherited autosomal dominant genetic disease where neuronal tissue degenerates. In this case, some symptoms may be noticed like jerky body movements and a decline in some mental abilities. By utilizing an immunoblotting assay as an initial screen, a 20-


mer, all G-oligonucleotide (HDG) has been identified as a potential therapeutic. A cell-based assay discovered that HDG is an effective inhibitor of aggregation of a fusion protein with the help of enhanced green fluorescent protein (EGFP) [32]. Another example for genetic disorders class of diseases is fabry disease, which is an X-linked disorder due to an insufficiency of alpha-galactosidase A and leads to the increase of globotriaosylceramide (Gb3) in various cells. Gb3 is a glycolipid accumulated in the blood vessels, and several other tissues and organs. The accumulation leads to an impairment of their proper function. The condition affects hemizygous males, as well as both heterozygous and homozygous females; males tend to experience the most severe clinical symptoms, while females vary from virtually no symptoms to those as serious as males. Immunofluorescence detection of Gb3 in conjunctival biopsies is a trustworthy method for the diagnosis of fabry disease, as well as to evaluate effectiveness of enzyme replacement therapy [33]. Presently, to understand the mechanism of bacterial diseases, several studies has been performed by using fluorescence. Tuberculosis is a common and often deadly contagious disease caused by mycobacteria, mainly Mycobacterium tuberculosis. Tuberculosis usually not only attacks the lungs but also affects the central nervous system, the lymphatic system, the circulatory system, the genitourinary system, the gastrointestinal system, bones, joints, and even the skin. Mycobacterium tuberculosis infection is a major clinical challenge today. A study has been conducted using fluorescein isothiocyanate (FITC)-labeled phosphorothioate ODN where M. tuberculosis is used to infect NR8383 cells. 1.5-7 fold increases in FITC-labeled phosphorothioate ODN accumulation have been measured by flow cytometry. It reveals that NR8383 cells that have been infected with M. tuberculosis can specifically accumulate ODN and this route of accumulation may lead to a means of drug targeting to mycobacteria-containing cells [34]. Recently, to determine the pathway of HIV-1 entry into human astrocytes and to know the fate of HIV-1 by detecting viral DNA, GFP-tagged HIV-1 has been employed [35]. Diabetes mellitus, often referred to simply as diabetes, is a syndrome of disordered metabolism, usually due to a combination of hereditary and environmental causes, resulting in abnormally high blood sugar levels (hyperglycemia) because of blood glucose levels are out of control by a complex interaction of multiple chemicals and hormones in the body, including the hormone insulin made in the beta cells of the pancreas. In other words, diabetes mellitus refers to the diseases that lead to high blood glucose levels due to defects in either insulin secretion or insulin action. In a study, transplantation of pancreatic islets has been performed to provide long-lasting insulin independence, a transplantation therapy for diabetic patients. These pancreatic islet-enriched fractions generate hormone-producing cells over 3-4 weeks of culture that has labeled, proliferating cells with a retrovirus-expressing green fluorescent protein. In this system, hormone-producing cells are generated de novo. This system can be useful as an experimental tool for investigating mechanisms for generating new islet cells along with GFP, and for designing strategies to generate


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proficient pancreatic islet cells ex vivo [36]. Several carbonic anhydrase (CA) isoforms are associated with plasma membranes. It is probable that these enzymes interact with anion transporters to facilitate the movement of HCO3- into or out of the cell. Multi Carbonic anhydrase isoform IX (CA IX) is highly over-expressed in many types of cancer and its expression is regulated by the HIF-1 transcription factor under hypoxia induction. Fluorescent inhibitors and membrane-impermeable sulfonamides have recently been used as proof-of-concept tools to demonstrate that CA IX is a potential target for anticancer drug development [37]. Heat shock protein 90 (HSP90) chaperones are over-expressed in some human cancers, which have prompted recent research on HSP90 inhibitors as new cancer therapeutics [38]. A fluorescence polarization assay has been developed to monitor the binding of compounds to the ATP-binding site of Hsp90 [39]. Thrombosis is the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system. To know the disease mechanism and pathophysiology of thrombosis, another multicausal disease [40], experimental models have been developed. Endothelial damage and clot formation have been monitored by fluorescent dye [41]. The mechanism of stent thrombosis has been evaluated by a flow chamber model where platelets were rendered fluorescently with mepacrine. Platelets thrombi formed around the implanted stents were continuously recorded by epi-fluorescent video-microscopy [42]. This fluorescent based research plays a major role in the search for therapeutics of thrombosis. DISEASE GENES BASED TARGET DISCOVERY Prior to the knowledge of DNA sequences of the human genome, disease genes have been identified based on hereditary patterns. These genetically inherited diseases run in families from an original initiator mutation; examples are like phenylketonuria, cystic fibrosis, Huntington disease, Fanconi’s anemia, and autosomal-dominant familial Alzheimer’s (FAD). The specific gene defects have been identified for a number of diseases that are the cause for hereditary disorder [43]. GFP, fluorescent microspheres, different other fluorescent dye and conformation-sensitive gel electrophoresis in fluorescent platform have been used to study the disease genes based target discovery. Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is progressive, which is genetically inherited neurodegenerative disorder caused by vasopressin (VP) gene mutations. Rats expressing an adFNDI VP transgene (Cys67stop) show a neuronal pathology, which is characterized by autophagic structures in the cell body using a fluorescent dye called monodansylcadaverine (MDC) [44]. MDC is a fluorescent dye that can be integrated selectively into autophagosomes and autolysosomes [45]. A rapid, costeffective readout method developed by Glaxo (USA) for single nucleotide polymorphism (SNP) genotyping, which combines an easily automatable single-tube allele-specific primer extension (ASPE) with an efficient high throughput flow cytometric analysis, has been performed on a Luminex 100 flow cytometer. An array of fluorescent microspheres has been hybridized to biotin-labeled ASPE reaction products, sequestering them for flow cytometric analysis [46]. This fluorescent microspheres based technique can


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provide large-scale human genotyping with a nominal number of steps, high accurateness and the ability to automate at a rational cost. Progress in DNA sequencing technology has enabled quick detection of disease genes through genetic screening. For target discovery associated with disease genes, DNA typing is very necessary. A new dimension for DNA typing analysis has been achieved with the development of technology for automated real-time analysis of fluorescent amplification products [47]. We can use this fluorescent DNA typing tool for disease genes identification, so-call gene based target discovery. A sensitive high-performance liquid chromatographic (HPLC) method for polymerase chain reaction (PCR) products using bisbenzimide (Hoechst 33258 dye) based fluorimetric detection has been developed, which has been also used to determined a point mutation. The genetic disease mutation like N-ras gene mutation could be detected specifically by this method [48]. N-ras gene mutations are caused to the aplastic anemia (AA) [49]. However, some diseases are not based on the mutation of a single gene, but a number of genes that altogether determine a person’s risk of developing a particular disease. For example, certain mutations in the BRCA gene family raise the risk for breast cancer. However, this risk does not always 100 percent certainties about the occurrence of the cancers. Conformation-sensitive gel electrophoresis (CSGE) has been developed as the most consistent method for the screening of multi-exon genes. The CSGE assay in a fluorescent platform has been developed to reduce the time of the assay and enhance resolving power for the complete scanning of the BRCA genes [50]. Mutations in BRCA1 and BRCA2 genes account for 5%-10% of familial early onset breast cancer. Currently, fluorogenic PCR is used to detect these genes. Identification of these mutations allows molecular diagnosis for breast cancer susceptibility. An automated PCR allelic discrimination assay (ADA) has been developed to identify the mutations in these genes [51]. Some allelic variants can increase susceptibility for diseases, such as the 4 alleles of apolipoprotein E (ApoE4) allele and 3 alleles of apolipoprotein E (ApoE3) for Alzheimer’s disease. The ApoE4 is a major risk factor for Alzheimer disease. So, ApoE may directly control neurons in the aging brain. EGFP-apoE reporter mice are useful for studying the regulation of apoE expression in the CNS, as well as the mechanisms of apoE4related neurodegeneration [52]. Using immunofluorescent and confocal microscopic localization, ApoE3 has been also detected as a risk factor for Alzheimer’s [53]. Thus, fluorescence-based techniques have showed very much competency for disease genes based target discovery. MAIN ‘TARGET DISCOVERY

CLASSES’

AND

TARGET

Presently, targets for therapeutic discovery can be broadly divided into five groups, like- receptors, proteins and enzymes, ion channel blockers, DNA, RNA and ribosomal targets [54]. Receptor Class For the study of receptor class, fluorescent proteins or probes are frequently used for experiments that are involved

for receptor site localization and expression using for drug candidates discovery. Presently, reporter-based assays are very popular tools for studying transcription, factors, gene expression, cell signaling path way, drug-target discovery. A number of reporters are available, which are genetically engineered or from natural sources like firefly luciferase, aequorin, obelin, GFP and their mutants [55]. The most important characteristics of these fluorescent reporters are: safe, easy to use, highly sensitive, wide dynamic response, and distinguishable from background. Receptors have been studied using radioactive isotopes or ELISA, and the disadvantages of these methods include radioactive hazards, heavy and awkward, and these limitations to study the molecular dynamics of receptor activation have delayed advancement in receptor research. Recent introduction of FRET and bioluminescence resonance energy transfer (BRET) has been proved as valuable tools in overcoming these disadvantages, and with the using of FRET and BRET, we can understand receptor dynamics without any hazards [2]. Some receptors, for example, the G-protein coupled receptors (GPCRs), have been successful used as therapeutic targets, and a number of drugs prescribed today have been discovered using this particular class. The GPCR target type is considered ‘drugable’ because they mediate cellular response to diverse cellular stimuli, including light, neurotransmitters, hormones, and odorants. For drug discovery, high throughput assays have been developed for the screening of ligands of these GPCRs [56,127]. A fusion of the receptor to the green fluorescent protein (GFPreceptor) has been expressed to investigate the before and after agonist exposure to receptor in suitable cell lines. Many GPCRs undergo ligand-dependent desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization into an intracellular endosomal compartment. GPCR-GFP-expressing cells are increasingly utilized in the study of such receptor dynamic events. One of such study has demonstrated the internalization of parathyroid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors [57,128]. Penn et al. [58] have reported a similar study with the h2-adrenergic receptor (h2-AR)-GFP conjugate expressed in HEK 293 cells. The procedure of GFP-tagging of the GPCR and its expression in a heterologous expression system has been followed by incubation of the transfected cells. The imaging of ligandinduced receptor internalization has been done in confocal microscopy coupled to digital image quantification that has been widely adopted for characterization of orphan receptors. Because GPCRs constitute widely accepted therapeutic targets, identification of their endogenous ligands has enormous potential for drug discovery. The GFP-based internalization assay provides a highly specific quantitative technique with sensitivity of nanomolar range. A unique heterodimerization pathway involving orphan receptors (TR2 and TR4) has been demonstrated by the intracellular localization of fusion receptors tagged with a GFP [59, 60]. Receptors from the members of the nuclearreceptor (NR) family, steroid hormones, thyroid hormones, and several vitamins have been shown by using GFP tags [61]. Chimeric GFP-receptors offer a new powerful tool to study the mechanisms of receptor translocation, to detect dynamic and


graded distributions of ligands in complex micro or macro environments, and to screen for novel ligands of “orphan” T receptors in vivo. GFP can be used as a fluorescent marker for total internal reflection fluorescence microscopy (TIRFM) [62]. This technique has enabled the direct observation of membrane fusion of synaptic vesicles and the movement of single molecules during signal transduction, shedding new light on important cellular processes near the plasma membrane [63]. Hence, GFP has proved to be a valuable tool in receptor research, thus considerably aiding the drug discovery process [64]. Activation of many of these GPCRs causes an elevation in Ca2+ levels. An automated highly sensitive assay is developed for calcium-coupled GPCRs using aequorin, another fluorescent molecule, as a reporter [65]. Other work has also reported a cell-based assay using aequorin as a reporter [66], which is an efficient tool for receptor research.

‘Proteins and Enzymes’ Class The second class is ‘proteins and enzymes’ class. This class also represent potential drug target for different diseases. A new research field has been developed to concentrate on protein function at the level of regulation of enzyme activity. This new area has been named as ‘chemical proteomics’, or ‘activity-based proteomics’, where the researchers use small molecules that can attach to catalytic residues in an enzyme active site. The chemically reactive group allows specific proteins or protein subsets to be tagged for purification and subsequently evaluate. As a result, this technology is able to identify new enzymatic proteins and has the potentiality to accelerate the discovery of new drug [67-72]. Targets of current interest include the enzymes involve in viral infection (e.g. HIV protease, influenza neuraminidase), glycoprotein and glycolipid processing (e.g. glycosyltransferases, sulfotransferases, glycosidases), inflammatory development (e.g. leukotriene A4 hydrolase), metastasis, and the biosynthesis of bacterial cell walls. Recently, another study concluded that abundant expression of leukotriene A4 hydrolase (LTA4H) and correlation with plaque instability identify LTA4H as a potential target for pharmacological intervention in treatment of human atherosclerosis where immunofluorescence staining is used, like Cy3( red fluorescent ) for 5-LO, FITC ( green fluorescent) for CD68, and DAPI(blue fluorescent) for DNA [73]. A group of novel activity-based fluorescent probes are designed and synthesized to target different protease subclasses based on their substrate specificities [74]. Cells contain numerous proteases, enzymes with proteolytic activities, which are found at different locations. These enzymes distinguish larger number of different substrates and are concerned in almost every process in the cell. Abnormalities in proteolysis are linked with several diseases, such as cancer, inflammation, arteriosclerosis, neurodegeneration and infection. Presently proteases are important targets for drug development due to their well-defined chemistry. Fluorescent based research has opened up enormous opportunities to visualize protease activities in the natural environment of the cells [75]. Recently, the first proteasome inhibitor, (‘bortezomib’; brand name ‘Velcade’) has been approved, which has been developed by Millennium (UK). This drug is a boronic acid inhibitor [76]


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and is indicated for multiple myeloma [76, 77]. Peptide degradation can also be observed using fluorophores. A strategy for detecting peptidase activity in living cells uses peptides containing FRET-fluorophore pair. FRET is operational with 10-100 Å and not detectable when fluorophores are more than 100 Å apart. So, spatial separation of two fluorophores has been made. As a result, protease activity can be easily assayed because it results to increase emission from the donor fluorophore at the expense of emission from the acceptor fluorophore [78].

‘Ion Channels’ Class The third target class is ‘ion channels’ class. Modulation of ion channel function is a successful area for drug development. Ion channel modulating drugs are used in the therapeutic treatment of epilepsy, hypertension, diabetes and chronic pain. Most of the ion channel-modulating drugs that marketed currently are developed without extensive knowledge of the molecular structure of ion channels, or the knowledge of how ion channel expression is regulated [79, 80,129,130]. Potential of new drugs can alter the function of a particular ion channel (in this case a K+ channel) by acting on accessory proteins that regulate the function of this channel. This can be achieved through the use of technologies and tools like LEPTICS® developed by Lectus Therapeutics (USA). It can be used to study interactions between ion channel proteins and their accessory proteins for development of new drugs [81]. Fluorescent tools have been used for this assay. Noncompetitive inhibitors (NCI) of acetylcholine receptor function include ion-channel blockers that obstruct the path used by cations to cross the membrane through the receptor channel. A single, high affinity NCI site has been identified by Omeros Corporation (USA) with multiple methods that are allosterically coupled to agonist binding. The microenvironment of this site has been studied using an environmentally sensitive fluorescent ligand. Analysis of fluorescence excited state lifetimes and spectral properties reveals that the inhibitor bound to the channel is highly immobilized and shielded from interactions with water. FRET method has also been employed using a variety of ligands to map the locations of the ion channel binding domain in the overall structure of the receptor [82]. Most of the early ion channel screen relies on the use of a fluorescent dye, and it is a very quick method. However, due to some compounds in ion channel, fluorescent properties can cause either false positive or negative results. Hydra Biosciences (Cambridge, MA, UK) has developed an assay that uses two different fluorescent dyes, because some potential compounds were accidentally discarded from an ion channel screen due to confounding effects of compound fluorescence. However, compatible fluorescent probes and functional assays are used to observe real-time target activity and to screen large chemical libraries for potent ion channel modulators. In fact, an article on Vertex Pharmaceuticals (USA) has addressed cell-compatible fluorescent probes, functional assays, and voltage/ion probe reader (VIPR) instrumentation that are used to monitor real-time target activity and screen large chemical libraries for potent and selective modulators [83]. It also discussed about advances and issues for both exogenously applied and fluorescent protein probes of cellular membrane potential, Ca2+, Cl-,


and pH. One familiar candidate in ion channel is the Kv1.3 potassium channel and it is of current interest for researchers due to its involvement in several clinically significant disease modalities. The voltage-gated potassium channel Kv1.3 is identified in human islet cells and T lymphocytes [84, 85]. Using membrane-potential sensitive florescent dye, a cell based assay has been developed for the identification of small-molecule antagonists against the Kv1.3 potassium ion channel [86]. Presently, fluorescent membrane potential probe kit is available and by using this kit, qualitative membrane potential assay has been developed and used to screen molecules for potassium channel closers (KCCs) and openers (KCOs) in insulin- and glucagon-secreting cell lines [87].

‘RNA’ Class The fourth target class is ‘RNA’ class. Pharmaceutical industry is paying attention on proteins, rather than nucleic acids, as drug targets. Recent advancement in the fields of RNA structure, synthesis, as well as target identification, makes us for the exploitation of RNA as a drug target. RNA has a link with genomics and bioinformatics, which also allows for increased discovery and specificity testing. There are many other advantages to targeting RNA, like sequencespecific binding, more sites accessible for interaction, and selective inhibition [88]. Several drugs that are targeting the rRNA of bacteria are available for clinical use at least for over half centuries. Aminoglycosides and macrolides are RNA-targeting antibiotics that inhibit prokaryotic translation through interaction with the A-site of ribosome. Sometime, targeting at the RNA level is an economical approach and RNA also offers entirely new opportunities for drug development, such as targeting of non-coding RNA sequences [89]. Fluorescently labeled oligonucleotide probes have been developed for evaluating the RNA affinity and specificity of HIV-1 Rev-RRE inhibitors that can assist in the effective targeting of therapeutically important RNA sites [90]. Fluorescence-based techniques are very much useful for probing to know the affinity between a RRE and small molecule and also its ability to inhibit Rev-RRE binding [91]. So, fluorescent probes and techniques are most convenient tool to detect therapeutically important RNA binding sites.

‘DNA’ Class The fifth target class is ‘DNA’ class. This class also represent potential drug target for different diseases. When DNA receives a signal, like in the form of a regulatory protein binding to a particular region of the DNA, it transcription or replication begins. Thus, if the binding specificity and strength of this regulatory protein can be mimicked by a small molecule, this molecule can act as therapeutic. So, synthetic or natural small molecule can act as a drug, if they can activate or inhibit the DNA function, although both these actions are possible to destroy cells for antitumor and antibiotic action. Drugs can bind with DNA by either covalent or non-covalent bonds. Covalent bonding leads to absolute inhibition of transcription or replication of DNA and eventually cell death. Cis-platin (cisdiamminedichloroplatinum) is a famous covalent binder used as an

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anticancer drug that binds covalently with the nitrogens on the DNA bases [92, 93]. Single and dual-labeled fluorescent oligodeoxynucleotides are used DNA based drug discovery, like to detect DNA-drug interaction. The effects of commonly fluorescent dyes and quenchers on the thermodynamic stability of a model probe-target DNA duplex have been investigated [94]. PHYSIOLOGY BASED DRUG DISCOVERY There are two different routes for drug discovery and development - one is target-based discovery and another is physiology based drug discovery. The difference between these two routes is the ‘time point’ for the invention of drug target. Physiology-based drug discovery follows physiological models which are created, for example, the disease phenotype is created in an animal model or cell-based assay and then compounds are screened through this readout. On the other hand, the process of target-based drug discovery starts with identification the therapeutic target and the function of the target for the creation of disease. A physiology-based strategy first starts with the screening of compound in an animal model and targets identification or validation follows in later stages [95]. A new hypothesis has been also proposed for drug discovery and development; that is based on pathophysiology and using animal models [96]. Several fluorescent tools and technologies have been used in animal models for drug discovery and development, like GFP, Red fluorescent protein (RFP), dual-color fluorescence imaging and fluorescent conjugated nanoperticale. Flow cytometry, fluorescent microscopy, confocal microscopy have been used to detect the florescence response. For detecting the different drug susceptibility of Leishmania, general techniques are very time-consuming, lengthy and require utilizing of macrophages. Therefore to monitor susceptibility of drugs to Leishmania, GFP and flow cytometry is used [97]. This fluorescent based model helps for the physiology-based drug discovery of Leishmania. At present, the chick-embryo model is an important model to study diseases like tumor growth, metastasis, angiogenesis and also for drug discovery. However, an image-able chickembryo model with a genetic fluorescent tag in the scattering cancer cells is developed. The combination of streptokinase and gemcitabine has been evaluated in this metastatic GFP chick-embryo model [98]. Zebrafish has become a widely used disease model. Mature zebrafish thrombocytes is the same as mammalian platelets [99]. Therefore, a zebrafish thrombocyte-specific cDNA library may provide a rich source of novel genes involved in platelet aggregation and blood clotting. An article from Zygogen (USA), a drug discovery company using zebrafish model, has been recently concluded that transgenic zebrafish with fluorescent thrombocytes makes the recovery of these cells and the synthesis of a thrombocyte-specific library straightforward [100]. Transgenic zebrafish with fluorescent blood vessels have been developed, where blood vessels are visualized very easily [101]. Compounds effects on blood vessel of this model can be seen very easily; therefore, the compound can be screened very simply. Several other transgenic fluorescent zebrafish diseases models have been developed [102, 103, 104]. These models can be used for compound screening.


Dual-color fluorescence imaging is a commanding tool to study cell biology in vivo model. Red fluorescent protein (RFP)-expressing tumors has been transplanted in GFPexpressing transgenic mice can be an excellent animal model for the cancer study has been suggested by AntiCancer Inc. (USA) [105]. The uses of the dual-color fluorescence cancer cells can be labeled in the cytoplasm and nucleus and associated fluorescent imaging provides an interesting tool to understand the mechanism of cancer cell migration. This model can help us physiology based drug discovery for anticancer drugs in mice model. At present, fluorescent conjugated nanoperticales are the powerful tools for drug discovery. Polyamidoamine dendrimers are conjugated to folic acid coupled to fluorescein or some other particle and conjugates are injected in a mice model having human KB tumors. These fluorescent conjugated nanoparticles targeting for anticancer drug improve therapeutic response [106]. An orthotopic metastatic mouse model for pancreatic cancer has been developed and the anticancer the efficacy of camptothecin analogue DX-8951f has been tested by AntiCancer, Inc. (USA). DX-8951f shows anticancer activities against two human pancreatic tumor cell lines. Cell lines are transduced with the green fluorescent protein, enabling highresolution visualization of tumor and metastatic growth. Hence, this fluorescent study supports anti-tumor activity of DX-8951f [107]. The recently generated GFP-reporter mice allow monitoring proteasome activities and the effects of proteasome inhibitors in vivo [108]. So, fluorescent tools are quite resourceful for physiology based drug discovery where chicken-embryo, zebrafish and mice models are used quite frequently. FUNCTIONAL DISCOVERY

GENOMICS

BASED

DRUG

Functional genomics brings mutually researchers from many different fields especially in chemistry, biochemistry, bioinformatics and molecular biology. Chemists, biochemist and bioinformatics experts join with molecular biologists to study the dynamics of the living cells. Their work relies heavily on tools and technologies adapted from the physical sciences as well as life sciences. Functional genomics took off with the creation of large gene expression databases. We are in the midst of a genomics revolution. With the completion of the Human Genome Project, we understand the sequencing of the entire human genome is about 100,000 genes or so in total. We have all those genes; to rationalize which ones we can look at for drug targets and other uses we have to understand function. The successful sequencing of the human genome has greatly increased the number of potential targets - the specific points for drug intervention in biochemical pathways. Pharmaceutical companies use such bioinformatics to screen for new targets at the first phase of drug development, so as to develop some of the compounds into drugs that affect the targets by blocking or enhancing a desired activity or function. As new proteins are identified and their activities determined through functional genomics, each of them will represent a potential new target for drug therapy. This attempt has been the catalyst for the genomics revolution and has necessitated the need to establish both new disciplines and new vocabularies like pharmacogenomics, genotyping, pharmacogenetics, micro-arrays,


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biochips, differential display, bioinformatics, chemoinformatics [109]. From the sequencing of the entire human genome, 700-800 GPCRs is estimated within the human genome, about 250 of GPCRs are identified as receptors for known ligands, and the rest are orphan receptors (OGPCRs). These OGPCRs are recognized as targets presently for drug discovery [110]. Functional GPCR-GFP (green fluorescent protein) fusion molecules are important tools for optical measurement of biochemical processes. With this tool, it has found that GPR120 and GLP-1 has potentiality as antidiabetic drugs [111]. With the help of functional genomics techniques, antibacterial drug discovery scenario has been changed paradigm shift from phenotypic screening to antibacterial to rational inhibition targets. Currently, antibacterial drug discovery is speed up by functional genomics that can improve our knowledge on gene functions for bacterial physiology and virulence or for the effects of antibiotics on bacterial metabolism [112, 113] where fluorescent tolls-like probes, GFP, and its variants are playing crucial roles. TARGET VALIDATION Target validation confirms the target that is involved in a disease process. With the target validation, we can also understand whether the modulation of the target can provide a therapeutic effect or not. Actually, in vitro and in vivo target validation is a proof of therapeutic concept and defines its clinical potential. Transgenic animal models, knockout animal models, and disease models are used for target validation. At the moment, small interfering RNA (siRNA) is an excellent tool for target validation. Recently, siRNAinduced down-regulation is analyzed by targeting a gene in EGFP transgenic mice that speed up the target validation for neuropsychiatric disorders that involve with a complex interplay of gene(s) from various brain regions [114]. Endometriosis is a widespread disease indicated by the estrogen-dependent ectopic growth of endometrial tissue. A model has been developed injecting a fluorescent fragments into nude mice; the growth of endometriotic-like lesions can be followed by fluorescent imaging. Significant decrease in lesion size has been noticed after use of Ganciclovir to treat animals implanted with human fluorescent endometrial fragments expressing thymidine kinase. This data designate that this animal model provides a tool for drug testing as well as gene target validation in endometriosis [115]. Antibody binding loops into four of the exposed loops at one end of green fluorescent protein (GFP) introduced as 'fluorobodies' have been developed and used effectively in ELISA, flow cytometry, immuno-fluorescence, arrays and gel shift assays, It has also shown affinities as high as antibodies. With the help of this properties, this 'fluorobodies' can use for high-throughput genomic scale selections, target validation and drug development [116]. Fluorophoreassisted light inactivation (FALI) uses coherent or diffuse light targeted by fluorescein-labeled probes which has been used in high-throughput methods for target validation [117]. Semiconductor quantum dots (QDs) are the rising fluorescent labels for imaging. For cellular imaging QDbased probes are very effective and this probe offers several advantages over organic dyes for target detection and validation [118]. An approach was developed related to


incorporation of green fluorescent protein (GFP) in the adenovirus vectors for efficient target functionality validation in vivo and in vitro [119]. It is likely that further development of these florescent based methods will improve the target validation. INFORMATION ABOUT FLUORESCENCE REAGENTS (GFP AND FLUOROPHORES) FOR DRUG DISCOVERY New generation of reagents especially fluorescent protein (FP) and organic fluorophores can report the specific molecular processes in living cells. Recent applications of fluorescent-protein and organic fluorophores have provided real-time assays for the analysis of molecular events in living cells which can help us immensely for the drug discovery process which we have already discussed in this paper. Fluorescent Proteins Fluorescent proteins from jellyfish and corals have revolutionized biological optical microscopy because they provide genetic encoding of strong visible fluorescence of a wide range of colors. Green fluorescent protein (GFP), a fluorescent protein, from bioluminescent jellyfish Aequorea victoria is widely known for its usefulness in biotechnology. Its unique ability to synthesize chromophore within itself, without any need for external substrates or cofactors except molecular oxygen, made it an excellent in vivo marker of gene expression and protein localization in various biological systems. GFP-like proteins are responsible not only for fluorescent colors in Anthozoa, but also for nonfluorescent purple-blue colors [120-121]. Other than purple-blue, cyan and yellowish-green emissions have been successfully generated from the Aequorea GFP4, none exhibit emission maxima longer than 529 nm1. Fortunately, the discovery of novel ‘GFP-like proteins’ from Anthozoa (coral animals) has significantly expanded the range of colours available for cell biological applications. As a result, the family of ‘GFP-like proteins’ deposited in sequence databases now includes approximately 30 significantly different members. Despite only a modest degree of sequence similarity, these GFP-like proteins probably share a -can fold structure that is central to the fluorescence of GFP. However, GFP and its variants have been used vastly in drug development process. At this moment, it is very apparent that GFP its variants are the major and significant candidates amongst the florescent tolls for drug discovery. The wider use of GFP [122,123] offers attractive options because of its noninvasive detection and ability to generate light independently of exogenous substrates. GFP and its variants can provide an opportunity for multicolored imaging therefore this tool can help us real-time imaging, especially when evaluating the efficacy of therapeutics [124]. In a word, GFP has revolutionized biology by enabling what was previously invisible. Extensive research in the development of GFPs ensures that in the coming years, facilitating studies of living systems will open-up possibilities for the clinical application of GFPs.

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Fluorophore Fluorophores are the analogy to a chromophore that also causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. Fluorescein isothiocyanate (FITC), a reactive derivative of fluorescein, has been one of the most common fluorophores chemically attached to other, nonfluorescent molecules to create new fluorescent molecules for a variety of applications. Other historically common fluorophores are derivatives of rhodamine (TRITC), coumarin, and cyanine.[2] Cy3 and Cy5 are reactive watersoluble fluorescent dyes of the cyanine dye family which are also routinely used in drug discovery. Newer generations of fluorophores such as the Alexa Fluors and the DyLight Fluors are generally more photostable, brighter, and less pHsensitive than other standard dyes of comparable excitation and emission [125]. These all fluorophores are regularly used in drug discovery process. Overall, the evolution of fluorescent-based tools and technologies within the context of drug discovery promises exciting possibilities. Presently, these technologies and tools are becoming key components for relieving the bottlenecks in drug-discovery process. As progress is made in automation, miniaturization, and in the discovery of new targets and their validation with the help of fluorescence techniques, new opportunities will soon materialize that will allow “drug candidates’ to enter into the next level rapidly. In future, fluorescent drugs will have the ultimate potential to show us more than what we actually want to know [126]. ACKNOWLEDGEMENT This work is particularly supported by "Aim for the Top University Plan" of the National Sun Yat-sen University and Ministry of Education, Taiwan. REFERENCES [1] [2]

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Lichtman JW, Angel CJ. Fluorescence microscopy. Nature Methods 2005; 2: 910-9. Arun KHS, Kaul CL, Ramarao P. Green fluorescent proteins in receptor research: An emerging tool for drug discovery. Journal of Pharmacol Toxicol Methods 2005; 51: 1-23. Deo SK, Daunert S. Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis. Fresenius J Anal Chem 2001; 369: 258-66. Collins LA, Torrero MN, Franzblau SG. A green fluorescent protein (GFP) microplate assay (GFPMA) for the high throughput screening of compounds against Mycobacterium tuberculosis was developed. Antimicrob Agents Chemother 1998; 42: 344-7. Changsen C, Franzblau SG, Palittapongarnpim P. Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter. Antimicrob Agents Chemother 2003; 47: 3682-7. Bergendahl V, Heyduk T, Burgess RR. Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase. Appl Environ Microbiol 2003; 69: 1492-8. Sandman KE, Marla SS, Zlokarnik G. Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and antitumor drug potential of platinum complexes. Chem Biol 1999; 6: 541-51. Gervaix A, West D, Leoni LM, Richman DD, Wong-Stall F, Corbeil J. A new reporter cell line to monitor HIV infection and drug susceptibility in vitro. Proc Natl Acad Sci USA 1997; 94: 4653-8.

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