emerging challenges in ligand discovery: new opportunities for chromatographic assay

Review

10.1586/14789450.2.6.891 © 2005 Future Drugs Ltd ISSN 1478-9450 891www.future-drugs.com

Emerging challenges in ligand discovery: new opportunities for chromatographic assay Ella Ng and David C Schriemer†

†Author for correspondenceUniversity of Calgary, SAMS Centre for Proteomics, Department of Biochemistry & Molecular Biology, Health Sciences Center, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, CanadaTel.: +1 403 210 3811Fax: +1 403 270 [email protected]

KEYWORDS: affinity chromatography, drug discovery, high-throughput screening, interaction analysis, ligand discovery, mass spectrometry

Ligand discovery initiatives are facing interesting challenges as ever-increasing numbers of proteins are entering screening programs. As an answer to steady pressure to improve performance in drug discovery, ligand discovery can expect to play an expanded role in generating small molecules as probes to help uncover the function of novel proteins. Chromatographic assay formats can offer new entry points into standard interaction characterization (binding and rate constants) as well as powerful, scaleable methods for compound screening. This review presents recent advancements in chromatographic assay technology, with a particular focus on frontal affinity chromatography as a platform technology for interaction analysis.

Expert Rev. Proteomics 2(6), 891–900 (2005)

The face of drug discovery initiatives willcontinue to evolve as technological advance-ments and socioeconomic realities are broughtto bear [1,2]. The science of genomics and pro-teomics continues to advance, causing theinevitable expansion of potential entry pointsinto disease management. Already, we canclaim that there are too few research resourceschasing too many drug targets, with thepharmaceutical market fragmenting away fromblockbusters [3]. Universities and research insti-tutes continue to increase their capacity to takebasic research to the next step, using innova-tive technologies leading to the opportunityfor drug discovery efforts to become broadlydisseminated through the public sector scien-tific community [2,4]. This is an essential evolu-tion of drug discovery as we seek progress inthe therapeutic assault on complex diseasesand degenerative conditions.

What are some of the technological implica-tions of this emerging reality, as it pertains totarget validation and early lead compound dis-covery? Will there be a measurable impact onthe techniques and methods that we use toevaluate the interaction of potential drugswith potential therapeutic targets? At thisstage, there is considerable debate over theimpact and ongoing value of high-throughput

screening in a drug discovery environment [5].It is clear that ligand discovery initiatives needto evolve, to better supply therapeuticresearch efforts. The ligand discovery and leaddevelopment phases continue to have ahealthy appetite for new biochemicalapproaches to ligand discovery and evaluation,even though the large pharma community hasalready invested heavily in technologies forinterrogating large libraries of moleculesagainst new targets. This review will presentsome of the current drivers for technologicalinnovation in ligand discovery and characteri-zation, and present an emphasis on the capa-bilities of biochromatographic techniques.Frontal affinity chromatography (FAC) willbe especially highlighted.

Ligand discovery & characterization in a postgenomic environmentInnovations in biotechnology have increasedour capability to both identify and expresspreviously unknown proteins, and thus thenumber of proteins that could be enrolledwithin a discovery program has expandedenormously. However, there is a critical lackof validation data to even suggest that suchproteins would be viable targets; completestructural and functional data on these new

CONTENTS

Ligand discovery & characterization in a postgenomic environment

Assessing the challenge for ligand discovery

Developing assays for ligand discovery & characterization

Potential of the chromatographic assay

Batch extractions in ligand discovery: affinity selection

Zonal methods in ligand discovery: affinity separations

Frontal methods in ligand discovery

Conclusions

Expert commentary & five-year view

Key issues

References

Affiliations

For reprint orders, please contact [email protected]

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892 Expert Rev. Proteomics 2(6), (2005)

proteins are usually lacking. The bottleneck in molecularbiologic research now sits at this functional level, creating theneed for new approaches to assess biologic behavior at themolecular level [6]. Techniques such as short interfering(si)RNA provide useful entry points to obtain functional data,but there is also considerable promise in the use of small-mole-cule ligands or inhibitors [7,8]. Indeed, they offer uniqueadvantages in that temporal regulation of function can be con-trolled, and individual domains of a multifunctional proteincan be specifically targeted.

As a result, the large-scale ligand discovery exercise hasextended beyond pure drug development initiatives, into thediscovery of probes or research-grade reagents for the purposeof functional elucidation. Appreciation of serendipitously dis-covered molecules such as colchicine, important for the earlydelineation of microtubule function, demonstrates how func-tional mechanisms may be elucidated via small molecules [9,10].The Molecular Libraries and Imaging Initiative of the NationalInstitutes of Health has been launched on this premise. Theinitiative is designed to implement high-throughput screeningof large compound libraries in a public-access model, for thepurpose of generating useful probes with which to test functionin cellular models. When in search of probe molecules, efficacycan receive the primary emphasis as the probe molecules are notoriginally intended to be drug leads.

Assessing the challenge for ligand discoveryThe decision to launch a screening exercise comes with a signifi-cant resource investment, leading to the conclusion that screen-ing may be applied prematurely if the protein does not becomea viable drug target. Cost-effective technologies for screeningneed to be advanced so that early ligand discovery is viewed asan opportunity for target validation and functional elucidation,not a follow-on activity in drug discovery. One response to thischallenge is to establish phenotypic cell assays, and capitalize onthe methods of chemical genomics [11,12]. An equally viableresponse is to enhance the ability of biochemical assays toaccommodate poorly characterized proteins and simplify thescreening exercise [13,14].

Implementing ligand discovery early in functional elucidationraises new challenges. First, there will be an increasedemphasis on ligand selectivity within a broadly defined pro-tein class and the requirement to screen compound collec-tions against numerous proteins [15,16]. This is needed inorder to derive a clear functional interpretation of phenotypewhen testing the newly discovered ligand(s) for function [17].Initially, this can result in a lowered reliance on function-based screening assays, but will eventually spawn a panel offunctional assays for assessing large numbers of hits. As aresult, discovery groups will need to maintain large andgrowing numbers of assays.

Second, greater efficiency in the use of available compounddiversity is required due to significant cost in compoundlibrary development and acquisition. Potent inhibitors maybe desirable, but at the early stages of functional elucidation,they are not an absolute requirement. As a result, assay hitscould be defined at a lower threshold (low-to-mid micromolardissociation constant; Kd) [18]. As long as selectivity is pre-served and compatibility with a cellular assay is retained,these can still be effective functional probes.

Third, the coverage of ligand space is very limited in anygiven compound collection, which suggests all compoundsources should be considered for screening: natural productextracts, combinatorial libraries and so on. Although com-pound collections may exceed 1 million compounds, thisnumber is not especially high, considering the theoretical esti-mates of molecular diversity [19]. Therefore, assays should beable to accept compound collections in a variety of formats(e.g., arrays, mixtures and crude extracts) to make maximumuse of available diversity [20–22].

While screening initiatives tend to be the domain of a smallnumber of well-outfitted laboratories, enabling technologiesfor both compound generation and library screening maymake these capabilities accessible to a larger number ofresearchers. A large number of corporations have emerged thatare willing to supply diverse collections of compounds toscreening groups. Indeed, the ability to create libraries ondemand through carefully prepared building blocks providesthe opportunity to proliferate focused libraries with greaterease [18,23]. Consideration will now be given as to whether assaytechnologies applied to ligand discovery can accommodate anexpanded role in functional elucidation.

Developing assays for ligand discovery & characterizationBiochemical assays of ligand binding or function (e.g., enzymeinhibition) only estimate true function within a biologic sys-tem, but they represent a useful simplification in the search forfunctional ligands. Over the past several years, standard plate-based assay technologies involving direct fluorescence, radio-activity or absorbance have been scaled to accommodate theincreased compound load [24]. Newer technologies have beenimplemented, including scintillation proximity assays, time-resolved fluorescence, fluorescence resonance energy transferand fluorescence polarization [25].

Figure 1. Negative-mode mass spectrum of a library (A) before and (B) after incubation with immobilized pepsin. In this example, the difference between the spectra identifies inhibitors to pepsin [30].m/z: Mass-to-charge ratio.

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Biochromatography in ligand discovery

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For laboratories seeking to add capacityor meet the challenges described above,there is little flexibility in current discov-ery systems to accommodate variety inlibrary formats, and increasing through-put in the number of proteins assayedwill be limited by the extensive assaydevelopment time. The power of array-based systems is undeniable, but thedependence on the quality of compoundcollections is high. As a result, this limitsthe compound diversity that can bescreened (and afforded).

Potential of the chromatographic assayChromatographic methods have becomeviable in the discovery and characteri-zation of molecular interactions. Funda-mentally, chromatography is designed todifferentiate between molecular specieson the basis of their adsorptive properties.When considered in a biochemical con-text, chromatography essentially repre-sents an assay in a column, withattributes similar to the existing biochemi-cal assay technologies [26]. Chromato-graphy can discover interactions in a sim-ple mix-and-read fashion. It can beautomated for medium-to-high through-put and can be used to collect thermodynamic and kineticbinding data. Chromatographic methods possess the addi-tional advantages of being precise, reusable and capable ofinterrogating impure mixtures or single compounds. As aresult, chromatography is less connected to the design andformat of the compound collection.

Historically, chromatography-based methods have sufferedfrom low sensitivity and extended assay development times, andwere unacceptably large scale, but this is no longer true. Theremainder of this article will describe advancements in the fieldof biochromatography, with a particular focus on FAC, and willseek to demonstrate the extensive assay design flexibility inher-ent to biochromatography. There is a broad range of chromato-graphic techniques available, and thus it is useful to categorizethese according to basic methodologic type:

• Batch mode/extraction: based on a single equilibration ofsample and chromatographic sorbent (the equivalent tomix-and-read technologies)

• Analytical chromatography: the classic chromatographicmethod where the sample is injected and separated on a column

• FAC: a continuous sample infusion mode, and the basis of thechromatographic biosensor

The author considers affinity electrophoresis to be outsidethe scope of this article and refers the reader to recent reviewson the topic [27].

Batch extractions in ligand discovery: affinity selectionAny equilibration of a compound with an immobilized orretained protein represents a batch-style chromatographicprocess, similar to that used in the fine chemicals industry forlarge-scale purification [28]. In this method, the compound ormixture of compounds is blended with an immobilized orentrapped protein and allowed to reach equilibrium (the statein which the concentration of bound compound and freecompound is constant). The next step is to separate boundcompound from free compound, for downstream identifica-tion and quantitation of the compound (usually by massspectrometry [MS]). One can either identify the capturedcompound [29] or monitor the depletion of compound(s) fromthe original mixture [30].

The latter approach is demonstrated in FIGURE 1, where animmobilized enzyme is used to extract ligands out of solution.This method requires careful analysis of the mixture pre- andpost-extraction, and may be difficult to apply to weak ligandsas the intensity changes will be slight. The method is quick andeasy once the protein immobilization procedure has been opti-mized. It suffers from a limited ability to characterize inter-actions thermodynamically and kinetically, and possesses diffi-culties with nonspecific adsorption to protein stationary phasesupports. A good survey of the variants to this technique canbe found in two recent reviews [31,32]. One of the moreadvanced examples of the batch-style approach is surface

Figure 2. Surface plasmon resonance mass spectrometry. A ligand is covalently attached to the surface of a SPR sensor chip and used for affinity capture of a protein. The protein can be analyzed by MS directly after washing (on-chip MALDI-TOF-MS) or after a recovery step [33].MALDI: Matrix-associated laser desorption/ionization; MS: Mass spectrometry; SPR: Surface plasmon resonance; TOF: Time-of-flight.

Polarizedlight Detector

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plasmon resonance (SPR)-MS, in which the biosensor chip ofa Biacore, Inc. instrument, for example, is equilibrated with acompound solution (FIGURE 2). The equilibration is monitoredvia the biosensor and, after a brief wash, the bound compoundis eluted into the mass spectrometer [33]. Most often, thismethod is applied to the discovery of protein ligands,although applications in small-molecule interaction analysisexist [34]. Since SPR-based instruments have lower limits ofdetection than MS techniques, it is usually necessary toincrease the amount of immobilized protein in order torecover more ligand. In doing so, the accuracy of the kineticdata may be compromised.

Zonal methods in ligand discovery: affinity separationsTraditional analytical (zonal) chromatography involves theapplication of a small-volume sample relative to a largercolumn, for the purposes of separating the solutes in the sam-ple. The old language of chromatographic plate theory is usefulhere; the more equilibration zones (plates) in a column, thegreater the efficiency in separation. In essence, the column rep-resents a series of batch extractions in a convenient format. Abiochemical assay can be developed in a column, where one ele-ment of the interaction is immobilized or otherwise entrained,thereby allowing for separation of the injected compounds onthe basis of affinity.

There are a number of benefits to crafting an assay in thisfashion. Columns can be regenerated so that the assay becomesstorable and reusable, much like an SPR chip [35]. This is advan-tageous when using a precious biochemical sample. The chro-matographic capacity factor of a ligand is a general property ofthe interaction and can be related back to thermodynamic bind-ing data [28]. In addition, the peak shape reflects the kinetics ofthe interaction [36]. An example is shown in FIGURE 3, where the

binding of verapamil to P-glycoprotein leads to peak broadeningrelative to a control [37]. This is expected for a slow tight-binding ligand. Other examples can be found in reviews byWainer [38] and Hage [26]. When incorporating specific markersor probe ligands, one generates a powerful method not only forsimple assessments of affinity, but also for interrogating thenature and mechanism of the interactions (e.g., competitive vs.noncompetitive inhibition) [39]. These methods have begun tomove well beyond proof-of-concept and into robust alternativesto the conventional assay. The enabling power of MS removesthe need for labeling, and researchers in this area have moved inthis direction. Perhaps most significantly, methods for columngeneration have advanced to the point where membrane-boundproteins can be introduced into the assay format [40]. As aresult, proteins from every general target class have beensuccessfully incorporated into chromatographic assays.

Nevertheless, a true homogenous, label-free assay formatwould be beneficial, where protein does not require immobili-zation and no labeling of a probe ligand is required. Theseefforts remain the limiting factor in chromatographic assaydesign. To meet this objective, size-exclusion chromatography(SEC) has been coupled with MS, which has proved useful forboth low-throughput characterization of interactions [41–43] andhigh-throughput screening exercises [44]. In this chromato-graphic assay, a compound or compound collection is pre-equilibrated with a target protein and then injected into a size-exclusion column for separation on the basis of hydrodynamicradius (FIGURE 4). In this situation, the protein and all boundcompounds will coelute early in the run, while the unboundcompounds will elute later. Capturing the early effluent fractionand analyzing it via MS supports the identification of hits fromlarge mixtures. There are kinetic limitations to this approach,where ligands with rapid off-rates go undetected; however, inmany situations this would only apply to weak ligands. The sizedifferential between protein and ligand must be sufficiently

Figure 3. Zonal affinity chromatographic profiles for a 3H-labeled verapamil solution (23.5 nM) through a chromatographic assay column (trace 3) containing membrane-bound transport protein P-glycoprotein. Traces 1 and 2 represent verapamil elution through a P-glycoprotein-negative control column, and a blank column containing only the stationary phase support, respectively [37].

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Figure 4. A homogeneous assay based on size-exclusion chromatography mass spectrometry technology. Assay development is simplified as no ligand labeling or protein immobilization is required [44].

Fast size-exclusion chromatography

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large to resolve in a SEC column, so this method would findapplication primarily in the discovery of low-molecular-weightcompounds targeting high-molecular-weight proteins.

An often overlooked advantage to all of these chromatographicassays involves the accessibility of the necessary infrastructure.Most research laboratories have access to high-performance liquidchromatography (LC) MS equipment that can be readily config-ured for assay purposes, requiring a minimum of additionalinvestment. This should support easy integration of the methodsfor the development of confirmatory assays in a drug discoveryenvironment. They should also be effective as secondary screens,where throughput is less of an issue, in the evaluation of hitsfor selectivity against a panel of related proteins and in molecu-lar models of general pharmacologic properties (adsorption,distribution, metabolism, excretion and toxicology).

Frontal methods in ligand discoveryPerhaps the most flexible and rugged chromatographicapproach for molecular interaction analysis and screening is themethod of frontal analysis. This unique style of analysisinvolves the continuous infusion of sample through a column,generating compound breakthrough curves rather than peaks.When conducted on bioaffinity columns, it is termed FAC [45].

FAC is an extremely useful addition to available technologiesfor thermodynamic and kinetic analysis of interactions. As withthe zonal technique, the discovery and characterization of amolecular interaction can be accessed in a concentration-independent manner in impure mixtures. This importantadvantage means that medicinal chemistry reaction products donot require rigorous quantitation or purification prior to affin-ity assessment, thus opening the door to accurate determina-tion of compound binding data from crude extracts or unopti-mized mixture syntheses (e.g., split and pool). The method iseven more simple to implement than zonal chromatography,and is capable of measuring a wider range of interactionstrengths (milli- to picomolar Kd).

FAC does not separate compounds, as shown in FIGURE 5, sothe detection of a specific binding event requires either the selec-tive labeling of a ligand and the use of a corresponding detector,or a detector capable of discriminating between coeluting com-pounds. If MS is applied as a detector, very complex mixturescan be resolved [46]. Although no physical separation of mixturecomponents occurs, the mass spectrometer provides gas-phasediscrimination, making generation of individual breakthroughcurves possible (FIGURE 6). Powerful modes for both low-through-put interaction analysis and high-throughput screening havebeen established, based on the FAC-MS combination [47–49]. Anintriguing application is shown in FIGURE 7 [50]. In this example,Slon-Usakiewicz and coworkers have shown that multiple bind-ing sites in a given protein target can be probed simultaneously,via the application of properly selected indicator ligands. Theauthors raise the interesting observation that in certain applica-tions (e.g., kinase ligand discovery), screening against theinactive or nonfunctional state may be more meaningful than afunctional screen.

The breakthrough curves generated by continuous infusionof sample ligand through an affinity column reflect the natureof the binding event under study. The position and shape ofthese curves depend on the thermodynamics and kinetics ofthe interaction, much as it does in conventional zonalchromatography [28,51], but breakthrough curves are easier todetect and data extraction is more straightforward in FAC.Models of varying degrees of complexity can be matched to thedata derived from breakthrough curves [52,53], and the interestedreader is referred to a number of articles outlining theapproaches available [35,54–56].

Some exciting new developments in FAC technology andmethods point to the growing power of this assay system. In gen-eral, affinity chromatographic methods require the immobiliza-tion of a protein or ligand, but the last few years have seen thedevelopment of entrapment technologies that can circumvent this(often difficult) step in preparing a chromatographic assay [57,58].

Figure 5. Schematic of the frontal affinity chromatography method. The equation relates measured breakthrough volumes for ligand (red) to nonligand (blue) and Bt.[A]0: Ligand concentration; Bt: Column binding capacity; C/C0: Normalized concentration; Kd: Dissociation constant; V - V0: Corrected breakthrough volume.

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Entrapment is achieved through the use of a protein-compatiblegelation procedure, involving silica-based monomers andadditives designed to preserve protein function and create aporous material. When cast into a solid, a material withchromatographic properties is created and, while not all pro-teins remain active, sufficient yield is often preserved to sup-port affinity chromatography (FIGURE 8). Brennan has recentlyapplied the entrapment process to the creation of FAC assaysfor interaction analysis of small-molecule ligands to dihydro-folate reductase, and has shown that the assays can be coupledwith either electrospray or matrix-assisted laser desorp-tion/ionization (MALDI)-MS techniques [59]. Simple andgentle entrapment procedures such as these offer a powerfulalternative to covalent immobilization, and shorten assaydevelopment time considerably.

It has recently been demonstrated that FAC can be operatedin a high-throughput mode, allowing for the discovery of lig-ands from mixtures exceeding 1000 compounds per analysis [60].It simultaneously overcomes the problems with online FAC-MS, namely the compatibility of high salt running buffersand electrospray MS. A glucosyltransferase FAC assay wasdeveloped to discover enzyme inhibitors from pools of modi-fied oligosaccharides [60]. Using a simple effluent fractionationscheme and a standard reverse-phase LC/MS configuration,it was shown that ligands could be identified and rank-ordered at rates greater than 5000 compounds per day. Theadded LC dimension in this FAC-LC/MS system provides

substantially greater sensitivity and larger mixture capacity thanthe previous online FAC-MS modes, and capitalizes on LC forbuffer removal. Increasing the speed of LC separation willreadily support screening rates approaching 50,000 compoundsper day.

ConclusionsBiochromatography offers considerable advantages to thediscovery community. New affinity formats and a wide rangeof accessible methods create a highly adaptable technology thatis only just beginning to make an impact on discovery initia-tives. Chromatographers have most often applied methods tostudy specific interactions and generate large-scale purificationsystems. Only recently has attention been turned to the genera-tion of highly miniaturized systems capable of sensitive analysisof molecular interactions. Generating the bioaffinity chromato-graphy column has always been the rate-limiting step in thedevelopment of a chromatographic assay, much as it is with thepopular SPR biosensor technology. SEC offers a solution to thisproblem, although it places limits on the range of kinetics thatcan be studied, and the molecular weight of the interactors.Through the adoption of entrapment technology, this time hasbeen reduced considerably and has further opened the door tothe preparation of whole-cell affinity constructs.

Bioaffinity columns can be processed using a number ofmodes, from the simple batch method to the more information-rich zonal and frontal methods, depending on the application.

Figure 6. Simultaneous frontal affinity mass spectrometry analysis of eight drug-like molecules against immobilized sorbitol dehydrogenase. Ligands spanning three orders of magnitude in binding strength could be resolved, demonstrating the ease with which ligands can be ranked [48].

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The frontal method is particularly powerful, in that it can supportsophisticated characterization studies and high-throughput ligandidentification modes. The adoption of MS as the detector ofchoice has been critical to the resurgence of chromatographicmethods in this field.

Expert commentary & five-year viewThe flexibility in experimental design and the more recentease of assay development will prove a useful addition in theevolving world of ligand discovery. Concentration-independent ligand screening and large mixture capacity areunique features of the general chromatographic method,making chromatography an important addition to the rangeof technologies available. This opens the door to the screen-ing of atypical sources of compound diversity; for example,natural product extracts and alternative combinatorial librar-ies [61]. The most immediate role for the chromatographicapproach is likely to be secondary screening exercises andligand characterization, where accurate and precise bindingdata are needed to sort the hits encountered in traditionalscreening exercises. Through chromatography, hits can bepooled and assessed in a single experiment. With thechromatographic assay’s ability to multiplex the number ofproteins screened, it should become economical to developprotein-class panels and apply them early on in the assessmentof selectivity.

Chromatography-based systems for ligand analysis remainresearch-grade and, while not complex, still require some opti-mization and adaptation to a particular problem. The next fewyears should see the proliferation of integrated systems betterdesigned for ligand analysis, incorporating nanofluidics andlab-on-a-chip technologies for heightened sensitivity. Whenthis occurs, one can expect a greater impact of the technologyon truly high-throughput screening exercises.

Large mixtures (>100 compounds) have been underutilizedas a source of compound diversity, partly because screeningtechnologies have not been easy to adapt. Chromatographicscreening methods should allow medicinal chemists to enablescreening exercises, through the single-pot preparation of size-able mixtures. These mixtures need not reflect a perfect synthe-sis; in fact, compound yield could vary over a wide range. Thisstems from the wide range in compound concentration tolera-ble to the chromatographic assay, and the concentration-independence in ligand evaluation. Looking further ahead,such screening capacity may become part of the medicinalchemist’s approach to library optimization: the syntheticmethod can be altered with a view to maximizing total ligandquality, not just the number of compounds.

One particularly interesting new application is fragment-based screening [62]. In this approach, a library is reduced to acollection of smaller components, each of which is screenedand the active components are used to assemble larger, morepotent inhibitors. As chromatographic assays excel in detect-ing weak interactions, even at low ligand concentrations,methods such as FAC should prove complementary to existingfragment-based screening practices.

What about the existence of related assay technologies? Thereare clear parallels between chromatographic methods and com-mercialized SPR-based biosensors (as popularized by Biacore,Inc.). Both can measure a wide range of ligand binding strengthsand share a common requirement of biomolecule immobili-zation. Label and label-free methods are supported and bothquantitative and qualitative binding studies of many differentinteraction types are possible. However, chromatography hasthe power to excel over SPR in small-molecule discovery initia-tives, as it offers the potential of greater sensitivity, screeningrates and compatibility with mixtures. This will prove very use-ful for screening novel proteins to discover functional probesfor downstream molecular biology.

Figure 7. Multisite competitive FAC assay of protein kinase Cα. Both the catalytic site and the ATP binding site of this kinase could be independently and simultaneously monitored. This example shows that an inhibitor peptide shifts the breakthrough curve of probe molecule for the catalytic site (purple), but not for the ATP site (pink). This example simultaneously highlights the indicator mode, the multiplicity screen and the benefits of independence from function [50].FAC: Frontal affinity chromatography; MS: Mass spectrometry.

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Figure 8. Protein entrapment by the sol-gel method. Reproduced with permission from the March 1999 cover of Applied Spectroscopy.

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ReferencesPapers of special note have been highlighted as:• of interest•• of considerable interest

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Key issues

• Ligand discovery initiatives are facing new challenges arising from an ever-increasing number of proteins to be screened. Specific pressures include genomics- and proteomics-driven target identification and early assessment of pharmacokinetic properties.

• Existing screening tools have limited flexibility, but high cost. The current screening paradigm is based upon assessing highly purified single-compound arrays against single proteins. Scaling the number of proteins screened, or including other sources of compound diversity, remain a challenge.

• Biochromatographic methods and systems offer solutions to the characterization of molecular interactions from complex samples. Main strengths include the ability to process mixtures, concentration-independent detection of ligands and scalability.

• Biochromatography has been applied to interaction analysis for all key classes of druggable proteins.

• Frontal affinity chromatography represents one of the most flexible formats for biochromatography, offering both sophisticated ligand characterization and high-throughput screening modes.

• Recent innovations in protein entrapment technology support true homogeneous assays based on the frontal affinity chromatography method.



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Ng & Schriemer


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Affiliations

• David C Schriemer, MSc, PhD

Assistant Professor & Director, University of Calgary, SAMS Centre for Proteomics, Department of Biochemistry & Molecular Biology, Health Sciences Center, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, CanadaTel.: +1 403 210 3811Fax: +1 403 270 [email protected]

• Ella Ng, MSc

University of Calgary, SAMS Centre for Proteomics, Department of Biochemistry & Molecular Biology, Health Sciences Center, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, CanadaTel.: +1 403 210 3811Fax: +1 403 270 [email protected]

emerging challenges in ligand discovery: new opportunities for chromatographic assay

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