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Arch. Pharm. Chem. Life Sci. 2007, 340, 169 – 177 S.-Y. Han and S.-H. Kim 169 Review Article Introduction to Chemical Proteomics for Drug Discovery and Development Sung-Young Han 1, 2 , Seong Hwan Kim 2 1 Department of Chemistry, University of Texas at Dallas, Richardson, TX, USA 2 Laboratory of Chemical Genomics, Bio-Organic Science Team, Drug Discovery Research Hub, Korea Research Institute of Chemical Technology, Daejeon, Korea A fundamental goal of chemical proteomics is to identify target proteins for bioactive small molecules and then apply them to drug discovery and development as valid and drugable tar- gets. Here, we introduce integrated technologies for the rapid identification of target proteins, methodologies for validating them as drugable targets, and applications of chemical proteomics in drug discovery and development. Keywords: Photoreactivity / Rational drug design / Structure elucidation / Received: September 19, 2006; accepted: February 1, 2007 DOI 10.1002/ardp.200600153 Introduction Chemical proteomics is a currently emerging field with a mission to identify and validate a target protein that directly binds with a binder such as a bioactive small molecule in a rapid, systematic, and comprehensive man- ner by design, synthesis, and application of relevant che- mical probes. As a multidisciplinary science, chemical proteomics proposes the integration of tools and technol- ogies from a variety of disciplines, chemistry, biochemis- try, biology, structural biology, proteomics, and infor- matics (Fig. 1). Thus, an integrated strategy in chemical proteomics can provide the information about the bind- ing site of bioactive small molecule in its target protein and, importantly, it could be the starting point to develop the drug screening systems based on the struc- ture of target protein. Mostly, the discovery of signal transduction pathway contributing to the disease state has been done in the first step of the processes for developing new drugs. The understanding of signaling pathways and further map- ping of the key signaling molecules in biochemical path- ways have provided how cells, organs, and the body are aberrantly controlled in disease state, and this had an enormous impact on the development of new therapeu- tic approaches for the treatment of nearly every human disease [1]. Thus, the efforts to reveal the signaling path- ways and molecules that are deeply involved in certain disease states have continuously developed the mechan- Correspondence: Seong Hwan Kim, Ph.D., Laboratory of Chemical Genomics, Bio-Organic Science Team, Drug Discovery Research Hub, Korea Research Institute of Chemical Technology, P. O. Box 107, Yu- seong-gu, Daejeon 305-600, Korea E-mail: [email protected] Fax: +82 42 861-0307 i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 1. Chemical proteomics as a multidisciplinary science.

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Page 1: Introduction to Chemical Proteomics for Drug Discovery and Development

Arch. Pharm. Chem. Life Sci. 2007, 340, 169 – 177 S.-Y. Han and S.-H. Kim 169

Review Article

Introduction to Chemical Proteomics for Drug Discovery andDevelopment

Sung-Young Han1, 2, Seong Hwan Kim2

1 Department of Chemistry, University of Texas at Dallas, Richardson, TX, USA2 Laboratory of Chemical Genomics, Bio-Organic Science Team, Drug Discovery Research Hub, Korea

Research Institute of Chemical Technology, Daejeon, Korea

A fundamental goal of chemical proteomics is to identify target proteins for bioactive smallmolecules and then apply them to drug discovery and development as valid and drugable tar-gets. Here, we introduce integrated technologies for the rapid identification of target proteins,methodologies for validating them as drugable targets, and applications of chemical proteomicsin drug discovery and development.

Keywords: Photoreactivity / Rational drug design / Structure elucidation /

Received: September 19, 2006; accepted: February 1, 2007

DOI 10.1002/ardp.200600153

Introduction

Chemical proteomics is a currently emerging field with amission to identify and validate a target protein thatdirectly binds with a binder such as a bioactive smallmolecule in a rapid, systematic, and comprehensive man-ner by design, synthesis, and application of relevant che-mical probes. As a multidisciplinary science, chemicalproteomics proposes the integration of tools and technol-ogies from a variety of disciplines, chemistry, biochemis-try, biology, structural biology, proteomics, and infor-matics (Fig. 1). Thus, an integrated strategy in chemicalproteomics can provide the information about the bind-ing site of bioactive small molecule in its target proteinand, importantly, it could be the starting point todevelop the drug screening systems based on the struc-ture of target protein.

Mostly, the discovery of signal transduction pathwaycontributing to the disease state has been done in thefirst step of the processes for developing new drugs. Theunderstanding of signaling pathways and further map-ping of the key signaling molecules in biochemical path- ways have provided how cells, organs, and the body are

aberrantly controlled in disease state, and this had anenormous impact on the development of new therapeu-tic approaches for the treatment of nearly every humandisease [1]. Thus, the efforts to reveal the signaling path-ways and molecules that are deeply involved in certaindisease states have continuously developed the mechan-

Correspondence: Seong Hwan Kim, Ph.D., Laboratory of ChemicalGenomics, Bio-Organic Science Team, Drug Discovery Research Hub,Korea Research Institute of Chemical Technology, P. O. Box 107, Yu-seong-gu, Daejeon 305-600, KoreaE-mail: [email protected]: +82 42 861-0307

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Chemical proteomics as a multidisciplinary science.

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170 S.-Y. Han and S.-H. Kim Arch. Pharm. Chem. Life Sci. 2007, 340, 169 –177

ism-based drug discovery, but it is not enough to developnew drugs for treating patients since the communicationamong the disease-related signaling pathways is toosophisticated to easily decipher the pathophysiologicalnetwork of the disease-specific signals.

Actually, as well as the understanding of the cross-reac-tion between a bioactive small molecule and its targetprotein(s) that can elucidate the biological actions ofboth, the structural information about candidate targetmay prove useful in ultimately predicting the interactionof a bioactive small molecule with its non-targeted pro-tein(s), which can lead to multiple side effects. Therefore,the structure-based drug design approach based on theunderstanding of ,what disease-related targets are’ in theoutset of the drug development process could cover theshortage of mechanism-based drug discovery includingunpredicted side effects.

As a rapid growth of structural biology for determiningthe protein structure and an advance in mass spectrome-try (MS) analysis, a structure-based drug design approachis considered a central component in the process of drugdiscovery and development. In fact, advances in processautomation and informatics have aided the developmentof high-throughput X-ray crystallography and the use ofthis technique for structure-based lead discovery [2, 3].Thus, virtual screening coupled with high-throughput X-ray crystallography is now focused on identifying one ormore binding small molecule fragments from chemicallibraries consisting of hundreds of small molecule frag-ments. The information about target proteins/peptidescan also make several other applications feasible. Two MS-based strategies, function-based and affinity-based, havebeen employed in recent years for screening and evalua-tion of compounds [4]. In the function-based approach,the effects of compounds on the biological activity of a tar-get molecule are measured, and in the affinity-basedapproach, compounds are screened based on their bind-ing affinities to target molecules. Therefore, the elucida-tion of interaction between a binder (e. g. substrate forenzyme, ligand for receptor, or bioactive small molecule)and its target protein(s) could be a promising way to speedup and further industrialize target-based drug discovery[5]. On the other hand, the efforts in chemical proteomicswould help to prioritize candidate targets, streamline thedrug development pipeline, and hopefully reduce thenumber of failures downstream.

With the fact, that chemical proteomics approacheshave gained more interests in the early stage of drug dis-covery and development together with chemical geno-mics that is to find and optimize chemical compoundsfor its use to directly test the therapeutic relevance ofnew targets revealed through genome sequencing [6], in

this review, the importance of chemical proteomics andseveral tools used in this field will be introduced and,finally, its application in the process of drug discoveryand development will be discussed briefly.

Impact of chemical proteomics

High-throughput screening assays used routinely is foridentifying a bioactive small molecule that can bind itstarget candidates and modulate their activities or func-tions, but a very large proportion of the hits identifiedare false positives that do not bind to the target bindingsite. This limitation can be overcome with the use of vali-dated target proteins that are identified and evaluated bychemical proteomics approaches.

Chemical proteomics study on the target proteins ofbioactive small molecules can also reveal alternative cel-lular modes of small molecule action, which was anunrecognized therapeutic potential previously. Forexample, pyrido[2,3-d]pyrimidines, which were initiallydeveloped as anti-proliferative agents targeting proteintyrosine kinases involved in cancer [7], were found tolack selectivity for tyrosine kinases. Instead, these inhibi-tors were established as potent inhibitors of several ser-ine/threonine kinases such as p38a and RIPK2. The identi-fication of alternative target proteins for bioactive smallmolecules can provide new insights into their cellularmodes of action. This could be highly associated with thefollowing; the identification of target proteins for anapproved medication by using chemical proteomicsapproaches play a pivotal role in the process of findingnew uses outside the scope of the original medical indica-tion for existing drugs that is known as redirecting,repurposing, repositioning, and reprofiling [8]. Reposi-tioning success stories and companies leveraging reposi-tioning strategies are increasing in number, but it shouldnot be discussed here.

To summarize, the elucidation of the specific interac-tion between bioactive small molecules and their part-ners, target proteins by using integrated chemical proteo-mics strategies contributes to our understanding of howbioactive small molecules work in cells and the body, andconsequently to the development of medicines.

Target proteins and binding sites

The biological function of most proteins depends ondirect and specific binding of their binders to particularspatial areas, the binding sites. Since the interaction ofbinders with target proteins affects their functional

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activities by changing the protein conformation, trigger-ing (or blocking) the protein post-translational modifica-tion such as phosphorylation or competing with sub-strates, the understanding of thermodynamics (or ener-getic stability) of interaction is important to elucidatethe biological activities of small molecules in cells.

Binding site (or pocket) can be defined as the region ofa protein that associates with a binder and it usually con-sists of a cavity in the protein surface formed by a particu-lar arrangement of amino acids. Binding depends on for-mation of contacts between chemical groups of a binderand those in the binding site of the target protein. In par-ticular, the cavity in the structure of a protein deter-mines the recognition features of the target protein.Thus, the characteristics of a binding site can provide themissing link needed for a thorough understanding of thecorrelation between chemistry space and genome space.

Recently, advanced and integrated methods for charac-terizing functional binding sites from a three-dimen-sional (3D) perspective have been developed and widelyused; the variety of methods using computer algorithmshave been continuously developed and can be commer-cially purchased from several companies such as AccelrysSoftware, Inc. (http://www.accelrys.com) and Tripos, Inc.(http://www.tripos.com). Using these methods, the virtualbinding sites of proteins can be identified or a suffi-ciently reliable homology model can be built [9], but themain limitation such as a high number of false positivescan not be excluded. This is the major bottleneck of insilico approaches.

In order to upgrade the quality of the protein models,the effort to improve the experimental methods such asX-ray crystallography and nuclear magnetic resonance(NMR) has been continuously made. The recent advancesin the automation of protein crystallization and in X-raydata collection/analysis have lead to the rapidly growingnumber of protein structures collected in the ProteinData Bank (PDB; http://www.pdb.org) [10]. Parallel withthis effort, the experimental identification and valida-tion of target proteins and/or binding sites by using inte-grated strategies in chemical proteomics should be alsoperformed since the precise elucidation of interactionbetween binders and their target candidates can help usto determine the crystal structure of proteins.

Technologies for purifying/enriching targetproteins/peptides

For elucidating the physical mode of action for bioactivesmall molecules identified by using cell-based, mechan-ism-based or effect-based screening system, it should be

considered first, how its binding partners can be purifiedfrom the complex proteome. Several approaches such asthe use of nanoparticle, photoaffinity probe, activity-based probe (ABP), and chemical affinity matrix havebeen developed to purify/enrich and identify target pro-teins/peptides that can directly interact with a bioactivesmall molecule [11–13].

NanoparticlesSeveral forms of nanoparticles have been used in biologi-cal separation. In particular, biomolecule-conjugatedgold nanoparticles (AuNPs) are the most popular probesbecause of their advantages such as the readily assem-bling with thiolated molecules, the large area/volumeratio for investigating 3D interactions, and the ease ofseparation by centrifugation [14, 15]. Recently, a newapproach of using carbohydrate-encapsulated AuNP hasbeen introduced [11]. This allows rapid mapping of carbo-hydrate-recognition peptide sequences in the target pro-teins.

Surface-modified magnetic nanoparticles can act as ageneral agent to separate, transport, and anchor a pro-tein with several advantages such as their high surface/volume ratio, good solubility, faster movement/easyentry into cells, and magnetically controllable aggrega-tion behavior that allow the intensive steps of centrifuga-tion omitted [16].

Photoaffinity probeMuch attention has been devoted to the application ofphotoaffinity labeling to the study of small molecule-tar-get protein interactions since photoaffinity techniquesare especially useful to elucidate the protein structure atthe interface of bioactive small molecules by photoche-mically labeling interacting sites [17–19].

Conceptually, the structure of photoaffinity probesconsists of four distinct functional elements; (a) a bioac-tive small molecule, (b) tags (such as biotin) for identify-ing and/or purifying the probe-target protein complex,(c) a linker region, and (d) a photoreactive moiety capableof covalently binding to target proteins (Fig. 2). Impor-tantly, the design of chemical probes runs parallel tostructure–activity relationships (SAR) study becauseprobes should retain the biological activity as its parentcompound does [20, 21]. Generally, the introduction of aphotoreactive moiety and a biotin into the structure of abioactive small molecule causes the decrease of biologi-cal activity, but probes with as much as 1000-times loweractivity can still be useful for investigating binder-pro-tein interactions [17].

Biotin is a powerful tag for purifying the probe-targetprotein/peptide complex based on the biotin-avidin inter-

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action that is the strongest non-covalent interaction(Kd = 10 – 15 M) [17, 18]. Biotin-tagged photoaffinity probescan be useful not only for the separation of the probe-tar-get protein / peptide complex in mixture (Fig. 3), but alsofor the indirect visualization of this complex by usingthe chemiluminescent reaction of horse-radish peroxi-dase conjugated avidin [22, 23].

The linker region between biotin, photoreactive moi-ety, and the bioactive small molecule can serve multiplepurposes; (a) to provide enough space to prevent sterichindrance that could decrease interactions between bin-der and active site of target protein in living cells, (b) toprobe the structural requirements for optimal biologicalactivity. In order to modulate the membrane permeabil-ity of a chemical probe in living cells and tissues, thealkyl linker spacer can be used. Polyethylene glycol (PEG)can also increase the bioavailability of probes by improv-ing its solubility in aqueous solutions.

Photoreactive moieties such as an azide, a diazirine,and a benzophenone group, have been used as precursorsfor the highly reactive intermediates, which are gener-ated upon photolysis [17–19]. Perfluorophenyl azide isthe most widely used photolabeling reagent since it canbe prepared easily. It belongs to a new class of photolabel-ing reagents with improved C-H insertion efficiency com-pared with nonfluorinated analogues. The fluorine sub-stitution on the ortho-position of an azide group can elim-inate the unfavorable ketenimine formation, which isless reactive with target proteins, and sufficiently extendthe lifetime of the highly reactive singlet nitrene, whichis important to obtain a stable covalent linkage. Trifluor-omethylaryldiazirine has been used as the photoreactive

group due to its many useful features; (a) good chemicalstability in various reaction conditions that may facili-tate the use of diazirines as carbene precursors; (b) photo-activatability under long-wavelength UV light (wave-length 350–360 nm) that minimize damage to pro-teins;(c) generation of highly reactive carbene that vir-tually reacts with proteins with no intramolecular rear-rangements. Benzophenone has been also used for photo-affinity-labeling experiments because of its photoactivat-ability at long wavelength (e. g. 365 nm). Benzophenonereacts preferentially with C-H bonds within 3.1 � of thecarbonyl oxygen and its high lipophilicity can be feasibleto modulate the hydrophobicity and cell permeability ofprobes.

Activity-based probe (ABP)The field ,activity-based proteomics’ makes use of ABP forprofiling the functional state of enzymes in complex pro-teome [24–27]. ABP generally consists of three basic ele-

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Figure 2. Structure of a biotin-tagged photoaffinity probe. A bio-tin-tagged photoaffinity probe consists of four distinct functionalelements, a bioactive small molecule, biotin, a linker region (e.g.PEG or alkyl), and a photoreactive moiety (e.g. azide, diazirine,or benzophenone).

Figure 3. Identification of target protein(s) by using biotin-taggedphotoaffinity probes. The UV-cross-linked complex of a biotin-tagged photoaffinity probe with target protein(s) can be purifiedand identified by using an avidin column and MS analysis,respectively.

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ments (Fig. 4); (a) an electrophilic reactive group (RG) thatis able to covalently attach to the active site of anenzyme, (b) a tag (e. g. biotin or fluorophore) that is usefulfor subsequent purification or visualization of probe-labeled enzymes, (c) a linker that connects these two ele-ments, provides selective binding interactions, and pre-vents steric congestion [28].

Based on a range of chemical scaffolds, ABPs have beendeveloped for more than a dozen enzyme classes includ-ing proteases, kinases, phosphatases, glycosidases, andoxidoreductases. The selectivity of a chemically reactivegroup in ABP allows specific proteins or protein subsetsto be tagged and purified or visualized (Fig. 5). As a result,the application of ABP is able to identify novel enzymaticproteins and characterize the mode of action of bioactivesmall molecule. The design and biological application ofABP has been reviewed in detail by Evans and Cravatt[29].

Two general strategies have been introduced for thedesign of ABP as shown in Fig. 4 [25, 26]. In the direct ABPapproach, well-characterized affinity labels can be incor-porated as the RG to direct probe reactivity towardenzymes sharing a similar catalytic mechanism and/orsubstrate specificity. This can be feasible to identify novelmembers of enzyme family to react with well-character-ized RG [30]. Whereas, in a second strategy referred tonon-direct ABP, libraries of candidate probes (biotiny-lated sulfonate ester in Fig. 4) are synthesized with vari-

able binding groups appended to a common RG, andthese reagents are screened against complex proteome toidentify activity-based labeling events. This method canfacilitate the identification of compounds possessingboth selective proteome reactivities and novel bioactiv-ities [31].

Chemical affinity matrixIn order to identify target protein(s) for a bioactive smallmolecule by using chemical affinity matrix, suitableattachment sites for immobilization should be predictedin a small molecule from SAR data obtained with collec-tions of structurally related compounds. However, in theabsence of any of such data, several potential attachmentsites can be selected to ensure that at least one of themenables the immobilization of a bioactive small moleculewithout conferring steric hindrance with respect to targetbinding through a systematic trial-and-error approach.

Small molecules possessing a functional group such asa solvent-exposed amino, carboxyl, or hydroxyl moietycan be covalently coupled to a suitable chromatographyresin; a variety of chromatography resins are commer-cially available that permit the immobilization of bioac-tive small molecules to the terminal functional groups ofhydrophilic spacer molecules. After verifying retentionof the biological activity, the small molecule-conjugatedchemical affinity resin can be utilized for the preparativepurification of target proteins from the relevant biologi-cal extracts [32–34].

Importantly, two kinds of chemical affinity matrices(positive and negative affinity matrices) should be runsimultaneously; despite of structural modification, posi-

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Figure 4. Two basic designs of ABP-based on the target selec-tivity. In the direct ABP approach, well-characterized affinitylabels can be incorporated as the reactive group (RG) to directprobe reactivity toward enzymes sharing a similar catalyticmechanism and/or substrate specificity, but in the non-directABP approach, libraries of candidate probes are synthesizedwith variable binding groups appended to a common RG.

Figure 5. Activity-based proteomics by using ABP. A typicalABP consisting of an electrophilic reactive group (RG, arrow)and a tag (fluorophore; circle) can be labeled with its targetenzyme(s) in a complex proteome and the activity-dependentlabeling event can be visualized by using in-gel fluorescencescanning.

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tive affinity matrix retains the significant activity andspecificity, but negative affinity matrix does not exhibitany potent activity [13]. This approach can differentiatepositive affinity matrix-specific binding proteins/pep-tides from those bound nonspecifically to both matrices(Fig. 6). Using affinity matrices, several proteins such asFK506-binding protein and mammalian histone deacety-lase 1 have been identified [35, 36].

Integration of liquid chromatography (LC) andMS analysis

The combination of LC and MS has had a significantimpact on drug development over the past decade. Con-tinual improvements in LC/MS interface technologiescombined with powerful features for structure analysishave resulted in a widened scope of application.

In general, the proteins that are enriched by using che-mical tools such as probes or affinity matrices can be

separated by gel (e. g. SDS-PAGE) and subsequently ana-lyzed by MS instruments. However, due to the exclusionlimitation in the sample preparation step and the lim-ited sensitivity of commonly used protein stainingreagents, the traditional separation method (e. g. two-dimensional gel electrophoresis) can not offer a lot ofinformation in the lower molecular weight fraction andthe low-abundance proteins of biological samples. Toovercome this problem, the enriched target proteins canbe digested with trypsin, and the resulting mixture isthen subjected to nanoflow reverse phase LC-MS/MS anal-ysis. Liquid-based separation can easily reach into thelower molecular weight protein and peptide range, anarea that is largely inaccessible to standard gel-based pro-teomics. Therefore, advanced and integrated technolo-gies such as electrospray ionization-MS/MS-integratednanoflow reverse phase HPLC for the separation/identifi-cation of peptide mixtures are now being increasinglyused in the field of chemical proteomics [34, 37]. Asshown in Fig. 6, target proteins can be captured by che-mical affinity matrices and after the following processesof washing, elution, and trypsin digestion, the compara-tive profiling of bound peptides allows rapid mapping ofpositive chemical probe-binding peptide sequences bynanoflow LC-MS/MS.

Consequently, the successful implementation of che-mical proteomics critically depends on not only the effi-ciency of affinity purification procedures, but also thesensitivity of MS analysis. For reference, the basic of MS,MS-based proteomics, and its applications (including tar-get identification and characterization, structure eluci-dation of synthetic compounds, and early drug metabo-lism and pharmacokinetics) in early stage of target-baseddrug discovery have been well-reviewed in the literatures[4, 38, 39].

Target validation by surface plasmonresonance (SPR) technology

To verify the specificity of the observed target proteins,simple experiments such as western blot analysis andcompetition binding assay were traditionally used[12, 23, 40]. However, these technologies do not providequantitative results of the relative sensitivities for thebioactive small molecule to the target protein and theseare not enough to turn out the initially identified pro-teins (or candidates for target proteins) to be a valid tar-get. Therefore, a need for target assessment that canprioritize a target candidate from an increasing numberof feasible targets being discovered through several tech-nologies is required.

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Figure 6. Identification of target protein(s) by using chemicalaffinity matrix. Target proteins can be captured by chemical affi-nity matrices and, after the following processes of washing, elu-tion and trypsin digestion, the comparative profiling of boundpeptides allows rapid sequence mapping of positive chemicalprobe-binding peptides (arrow-indicated) by LC-MS/MS.

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Physicochemical properties for molecules binding toits target protein affect the biological function of targetprotein. Thus, the data showing the kinetics betweensmall molecules and proteins could be particularly help-ful in target assessment.

Without any labeling, SPR imaging permits monitor-ing of biomolecular interactions by detecting the inten-sity change of reflected lights resulting from changes inthe refractive index on the gold surface [41, 42]; thechange in mass concentration at the surface can bedetected in real time with data presented as a sensor-gram (SPR response plotted against time), which displaysthe association and dissociation of complexes with thekinetics (Fig. 7). However, before examining the kinetics,the purified or recombinant target proteins should beprepared and then immobilized onto the sensor surfaceas an interaction partner. Immobilization occurs bydirect coupling to the surface or via a capture moleculecoupled to the surface.

Recent SPR instruments have improved sensitivity andsample capacity, and better tools for sample recovery andthe interface with MS. Therefore, the kinetics betweensmall molecules and target proteins can be simulta-neously analyzed and/or those with high affinity and spe-cificity to the target can be directly identified in the fol-lowing MS analysis.

Applications of chemical proteomics

Methods for chemical probes can provide a new avenuenot only for identifying novel target proteins (target dis-

covery), but also for identifying/evaluating chemical inhi-bitors thereof (inhibitor discovery) as shown in Fig. 8 [26].

The identification and functional annotation of therelevant binding site(s) in a target protein for a bioactivesmall molecule can provide new strategies in the processof drug discovery and development such as structure-based drug design and lead optimization [24]. Usingdirect binding SPR assays, compounds can be rapidlyidentified to be with specificity in the binding site of avalidated target protein or candidates. For example, thekinetic data obtained from SPR biosensors has been usedin the screening of kinase inhibitors or potential priondisease therapeutics [43, 44]. After compounds screened,the approach to find and optimize the lead compoundscan be followed [45]. A new strategy for improved second-ary screening and lead optimization using high-resolu-tion SPR characterization of compound-target interac-tions has been recently reviewed [46]. In this report, thestructure-based biophysical analysis method integratingSPR with bioinformatic approaches and mutation studiesin the early drug discovery process suggested to be usedas a cost-effective alternative to high-throughput screen-ing methods in cases when detailed binding site informa-tion is available. Binding site-modified proteins can beused as reference targets in direct binding SPR assaysaimed at identifying compounds with specificity to bind-ing site of real target protein.

In addition, ABP per se has recently been shown to havea great potential to facilitate the process of lead drugidentification [24]; for example, of hypothetical leaddrug candidates, the lead drug can show the most specifi-

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Figure 7. SPR sensorgram presenting SPR response (or reso-nance signal) plotted against time. The sensorgram displays theassociation and dissociation of complexes over the entire courseof an interaction with the kinetics.

Figure 8. Chemical proteomics and its applications in drug dis-covery and development.

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city for the intended drug target in a competition bind-ing assay with the chemical probe.

Together with secondary screening and lead optimiza-tion, SPR technology can also be applied in a variety offields including activity characterization, compound spe-cificity studies, early in vitro ADMET (adsorption, distribu-tion, metabolism, excretion, and toxicity) evaluation andquantitative SAR analysis [45]. In clinical analyses such asimmunogenic study and food industry, the presence ofpathogens, toxins, veterinary drugs, and nutritionaladditives in food samples is being investigated with SPRbiosensors [47, 48].

The information about the binding site or 3D structureof target proteins that is extracted from chemical proteo-mics approaches can be used for providing virtual hits byin silico screening of large substance library. Virtualscreening methods have increasingly been used as a com-plementary means of finding small molecules that areactive with a particular target. In this screening, detailedknowledge about the binding site of the target proteinprovides a promising starting point for finding com-pounds with selectivity that are suitable as lead candi-dates and their biological activity can be directly evalu-ated in in-vivo studies.

Consequently, the contribution of chemical proteo-mics to the pipeline in drug discovery and developmentwith its several applications are being deployed in newways to boost the efficiency of discovery and develop-ment, the quality of compounds moving through thepipeline, and the speed with which higher-quality drugcandidates enter into the clinic.

Conclusion

It is very clear that the success in the development of newsmall molecule therapeutics could be decided in the stepto choose a suitable target because a bad choice makes uswaste time and money in competitive drug markets. Indrug discovery and development, chemical proteomicsstrongly presents the possibility of speeding up targetidentification and getting higher-quality leads in a rapid,systematic, and comprehensive manner by design, synth-esis, and application of relevant chemical probes. In thispromising approach, the interplay of chemistry and biol-ogy drives target selection and lead identification withintegrated strategy; chemistry can provide the criticaltechnologies in the step to design and synthesize the che-mical tools, and then, the advanced disciplines such ascell biology, biochemistry, proteomics, structural biol-ogy, bioinformatics, and physical chemistry provide pro-found knowledge of compound selectivity and its interac-

tion with target proteins. Importantly, the elucidation ofthe relevant binding site(s) in a target protein for a bioac-tive small molecule can provide new strategic applica-tions for lead finding, lead optimization, and develop-ment of structure-based drug screening system.

In conclusion, together with technologies developedin a variety of disciplines and improved chemical and bio-logical information provided from websites (e. g. ProusScience Integrity, http://www.prous.com; Discovery Gate,http://www.discoverygate.com), the advances in chemicalproteomics and its expansion in drug discovery anddevelopment could provide new potential for efficientdiscovery of new classes of drugs in the near future.

SHK was supported by ,Chemical Genomics Research Project‘,Korea Research Institute of Chemical Technology, Republic ofKorea and ,Chemical Genomics R&D Project‘, Ministry ofScience and Technology, Republic of Korea.

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