Afnity selection-mass spectrometry screening techniques for small molecule drug discovery

D Allen Annis, Elliot Nickbarg, Xianshu Yang, Michael R Ziebell and Charles E Whitehurst
Afnity selection-mass spectrometry (AS-MS) techniques assess the binding of candidate molecules to immobilized or soluble receptors, and these methods are gaining acceptance in high throughput screening laboratories as valuable complements to traditional drug discovery technologies. A diversity of receptor types have been evaluated by AS-MS, including those that are difcult to screen using traditional biochemical approaches. AS-MS techniques that couple liquid chromatography-MS with size-based separation methods, such as ultraltration, gel permeation, or size-exclusion chromatography, are particularly amenable to the demands of MS-based screening and have demonstrated the greatest success across a broad range of drug targets. MS measurements of receptor function have many of the same advantages as AS-MS screening and are increasingly used for drug discovery as well.
Addresses Schering-Plough Research Institute, 320 Bent Street, Cambridge, MA 02139, United States Corresponding author: Annis, D. Allen (allen@allenannis.com), Nickbarg, Elliot (elliot.nickbarg@spcorp.com), Yang, Xianshu (xianshu.yang@spcorp.com), Ziebell, Michael R. (michael.ziebell@spcorp.com) and Whitehurst, Charles E. (charles.whitehurst@spcorp.com)

use MS as the detection system are of particular interest because of the exquisite sensitivity and unique selectivity possible with MS. In some screening formats, the selectivity achieved by MS enables direct analysis of compound mixtures such as combinatorial libraries and unpuried natural products extracts an advantage that is not easily achieved using other analytical methods. MS-based screening is primarily implemented in two ways: by monitoring the functional output of a receptor-dependent biochemical reaction, or by using afnity-based methods that directly assess binding of a candidate molecule to its target receptor. Because of their expanding popularity, both of these styles of MS-based drug discovery have been the subject of several timely reviews, special issues of topical journals, and the focus of at least two recent books [2,3,47]. Herein we report on the recent contributions to the eld of MS-based drug discovery, with special emphasis on lead identication methods that couple an afnity selection step with MS conrmation of receptorligand binding.

Afnity selection-MS: direct detection of receptorligand complexes

Afnity selection-MS (AS-MS) methods directly or indirectly measure binding of small molecules to their biomolecular target, and all varieties of AS-MS include the following steps: (i) an afnity selection stage, where the protein is equilibrated with one or more potential ligands, allowing the protein to form a complex with any compound capable of binding; (ii) the resulting receptor ligand complexes are separated from non-binding mixture components; and (iii) ligands are identied by MS or MSMS. Direct AS-MS methods (Table 1) separate proteinligand complexes from unbound components within the mass spectrometer, then measure the mass of the non-covalent or (less commonly) the covalent proteinligand complex in the gas phase. Indirect methods (Table 2) use a separation technique, typically chromatographic in nature, to isolate the proteinligand complex from unbound components before MS analysis. Both direct and indirect varieties of AS-MS have been developed in an assortment of hardware congurations and successfully applied to screening compound mixtures, including combinatorial libraries and unpuried natural products extracts. Researchers at Sunesis developed a fragment-based, direct AS-MS technique to identify compounds covalently bound
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Current Opinion in Chemical Biology 2007, 11:518526 This review comes from a themed issue on Analytical Techniques Edited by Peter M Fischer

1367-5931/$ see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.cbpa.2007.07.011

Introduction
Mass spectrometry (MS) is one of the most powerful analytical techniques in modern science, and it plays a key role in nearly every stage of the drug development process [1]. Recent advances in MS instrument design, especially the development of electrospray ionization (ESI), have extended the reach of MS to the lead discovery stages of drug development as well. Modern pharmaceutical discovery relies increasingly on target-based screening techniques to identify new lead compounds for receptors that have known or suspected involvement in a disease pathway. High-throughput screening (HTS) methods that
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Affinity selection-mass spectrometry screening techniques for small molecule drug discovery Annis et al. 519

Table 1 Direct AS-MS screening approaches Class Covalent complexes Method Fragment-based lead assembly Description Ligand fragments bind covalently to the target and are identied by MS Gentle ionization technique enables covalent complexes to be preserved in the gas phase Advantages Fragment library permits interrogation of a large portion of chemical diversity space High sensitivity permits low compound library concentrations. A high density chip at the ESI source increases throughput. Small sample volumes Multiple targets may be screened in the same sample. Potential to identify low afnity cmpds missed by other approaches Used as counter-screen to identify ligands with high afnity to DNA. Automated sample prep permits >1000 samples/week Generates data on contact sites as well as putative mechanisms of binding. Appropriate for ligand optimization efforts Disadvantages Requires a reactive side chain in vicinity of binding site. Assembled fragments may not generate active compounds Small molecule library size is limited and HTS not yet demonstrated. Gas phase measurements may not reect biological interactions Mass envelope of ligand-target complex may not be observable by spectrometer

Noncovalent complexes

Nano-electrospray-MS

Multi-target afnity/ specicity screening (MASS)

Direct MS detection of noncovalent RNA-ligand complexes

Detection of oligonucleotide-ligand complexes by ESI-MS (DOLCE-MS) ESI-electron capture dissociation

Direct MS detection of ligands bound to single or double stranded oligonucleotides The sites of ligand-target contacts may be investigated using ECD for complexes that remain intact in the gas phase

No information on specic oligonucleotide sites of interaction

Useful primarily for high afnity interactions. May not work for large complexes where conformation in the gas phase is altered

to a target protein [8,9]. In their site-directed ligand discovery approach, specic amino acids on a protein surface are modied to facilitate tethering of small molecule building blocks (fragments). The protein guides fragment assembly within a binding pocket to select those building blocks with the best binding characteristics. The resulting covalent proteinligand complexes are directly analyzed by MS, and the measured shift in molecular mass indicates which building blocks yield the best ligand. Medicinal chemistry efforts, often in conjunction with X-ray crystallographic analysis, are subsequently employed to convert the covalently bound fragments into a noncovalent ligand with better afnity and molecular properties than the progenitor fragments. AS-MS techniques that directly interrogate noncovalent proteinligand complexes are of considerable value because the resulting leads are inherently more drug-like than those arising from covalent approaches. These methods require that the non-covalent complexes survive the transition to the gas phase, and in 1991 Ganem and Henion presented the rst conclusive evidence that the immunosuppressant FK506 and its biomolecule target FKBP can be directly observed as a gas-phase complex using ESI-MS as a gentle ionization technique [10]. Since this seminal report, many similar non-covalent receptorligand interactions have been identied and characterized using ultra-sensitive nanospray ESI-MS [11,12]. An excellent review was
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recently published by Hofstadter and co-workers on the detection of small molecule DNA and molecule RNA binding by multi-target afnity/specicity screening, or multi-target afnity/specicity screening (MASS) [13]. Zhang and co-workers have commercialized a chip-based ESI-MS system and demonstrated its utility for studying proteinligand interactions [14]. An especially useful feature of this system is its ability to quantify binding afnities using a minimal amount of puried receptor [15]. As an added benet, Loo and co-workers have shown that electron capture dissociation analysis of the proteinligand complex inside a Fourier transform ion cyclotron mass spectrometer can reveal the binding site of a small molecule on its receptor [16]. ESI-MS methods for detecting receptorligand binding are appealing because they give direct evidence of the existence of a complex. However, conicting data on the correlation of gas-phase afnity measurements with solution-phase interactions have been shown [17], including reports by researchers using a new, laminar ow technique that estimates receptorligand afnities by ESI-MS while not requiring that non-covalent interactions are preserved in the gas phase [18,19]. Also, these and other related direct AS-MS techniques do not tolerate high concentrations of non-volatile salts, buffers, cofactors, metal ions, or detergents that may be necessary for proper protein folding and stability.
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Table 2 Indirect AS-MS screening approaches Class Size exclusion chromatography and gel permeation chromatography Method Automated ligand identication system (ALIS) SpeedScreen Description Target-ligand complex captured by fast sec followed by integrated LCMS ligand identication Target-ligand separation using 96-well SEC plates followed by LCMS Compounds infused through a column having a target immobilized on the solid phase. Weak binders elute early, strong binders elute later Compounds combined with bead-bound target followed by washing, elution and detection by ES-MS/MS Target-ligand complexes are separated from other components by electrophoretic mobility and bound ligands identied by MS Enrichment of target-ligand complexes by pressure-based ultraltration, then ligand dissociation & detection by ESI-MS Ligand-target complex retained on membrane is extracted and ligands are dissociated and identied by ow-injection MS Advantages Large mixtures permit HTS. >10 000 compounds screened per hour per instrument 96-well plates permit HTS (400 000 cmpds/week) when screened in pools of 400 compounds Generates afnity ranking for binders. Controls for differences in MS sensitivity between cmpds. (10 000 compounds/day) Permits interrogation of protein complexes as well as poorly solubilized receptors Flexible assay format. Potentially useful for a range of targets. Short sample runtimes Large library mixtures are possible increasing the throughput to >200 000 compounds in 2 months Applicable for targets with poor chromatography characteristics when tried with other separation methods Disadvantages Samples are processed linearly (6 min/sample) limiting HTS of individual compounds Total organic content in bed volume is limited. Receptor-library separation is not directly visualized Protein must be modied for attachment. Library size is limited by elution overlap and competition Protein must be modied for attachment. Background possible due to nonspecic binding Ligand must modulate the target electrophoretic mobility. Requires volatile solvents to accommodate MS Multiple steps and incomplete separation of bound and free ligands and possibility of background noise Ligand binding may be disrupted by target interactions with the membrane. Washing required

Solid phaseimmobilized target

Frontal afnity chromatography with MS detection

Afnity capture-MS

Afnity capillary electrophoresis-MS

Separation by ultraltration

Ultraltration-MS

Pulsed ultraltration-MS

Afnity selection-MS: indirect (hyphenated) methods

Immobilized ligand or receptor

In part to enable the use of non-volatile buffers in the receptorligand binding reaction, indirect AS-MS screening methods have been developed that couple a separation technique with ESI-MS detection. The simplest variants are those where one binding partner, either the receptor or the small molecule ligand, is immobilized on a solid support. Familiar non-MS methods infer receptor ligand interactions by measuring changes in surface plasmon resonance (Biacore) or wavelength shift (Cornings EPIC and SRU BioSystems BIND platforms) [20,21]. MS-based techniques detect ligands that have been captured by an immobilized receptor; for example, Schriemer and co-workers have developed a frontal afnity chromatography (FAC)-MS platform for lead discovery. In FAC-MS, a sample containing a set of ligands and a non-binding marker compound are passed through a column onto which a receptor has been attached. The non-binding marker compounds elutes in the void volume, while each ligand elutes at a later time depending on its afnity for the immobilized receptor [22,23].
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Brennan and co-workers have immobilized nicotinic acetylcholine receptors on monolithic FAC columns and conrmed the activity of the bound receptor using epibatidine as control ligand. In this manner they used FAC-MS to optimize protein immobilization methods for on-line enzyme assays [24,25]. Researchers at Protana have also reported FAC-MS as a global kinase-binding assay with a throughput of up to 10 000 compounds per day [26]. They demonstrated their method by discovering inhibitors of the EphB2 tyrosine kinase receptor, and described other applications of the platform in a recent review [27,28] Afnity capillary electrophoresis (ACE) is a well-established technique for drug discovery from complex mixtures, including natural products [29]. Despite improved sensitivity and selectivity by using MS as a detection system for ACE, it requires the use of volatile buffers in order to interface with MS, so this application is not routinely used [30]. Afnity chromatography techniques, including ACE-MS and FAC-MS, work well for analyzing compound mixtures,
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Affinity selection-mass spectrometry screening techniques for small molecule drug discovery Annis et al. 521

and can also yield quantitative binding afnity estimates for a variety of receptor types. Additionally, the afnity columns can be reused. However, these methods require that the receptor be immobilized, which can affect its binding behavior relative to the native, soluble target, and may cause false positives from compounds binding non-specically to the stationary phase or linker system. While direct MS analysis of the afnity column is challenged by nonvolatile buffers, integration with matrixassisted laser desorption/ionization mass spectrometry (MALDI-MS) may overcome this limitation [31].
Solution-based methods

these latter two reports are interesting examples of ultraltration-based AS-MS, as described these methodologies are not immediately amenable to HTS. Gel permeation chromatography (GPC) spin columns are also used to separate receptorligand complexes from unbound ligands for AS-MS, and Siegel recently provided a detailed review of this approach as practiced by Amgen, Novartis, and Wyeth Pharmaceuticals [41]. A limitation of this approach is that spin-column handling and centrifugation may be more difcult to automate and scale than other methods; however, overall throughput can be increased by pooling the eluants from several spin columns before ESI-MS analysis. Mayr recently reviewed the successful use of SpeedScreen, an AS-MS method developed at Novartis that uses a centrifuged 96-well plate assembly of GPC columns, including a top sample loading plate with pinholes in the bottom of each well; a central lter plate pre-loaded with GPC resin (Sephadex, Pharmacia); and a bottom collection plate for the capture of efuents containing separated receptorligand complexes [42]. Brown et al. reported on the chemical nature of hits identied from the SpeedScreen-based HTS of 26 targets [43]. They concluded that SpeedScreen yields compounds with drug-like properties, albeit with greater lipophilicity and higher molecular weights than the progenitor compound screening library. The authors emphasized the value of using cheminformatic-based lters to eliminate promiscuous binders, false positives, and disfavored chemotypes from primary hit lists, and further emphasized the importance of hit validation by rigorous follow-up SpeedScreen assays. Size exclusion chromatography (SEC) separates protein ligand complexes from non-binding small molecules by the same principles as GPC. However, SEC holds several advantages relative to the spin-column method, including its amenability to automation and the ability to directly couple the SEC stage with an LCMS system for ligand detection. The rst fully integrated SEC-LCMS platform for HTS was developed by researchers at NeoGenesis (later acquired by Schering-Plough) and dubbed the automated ligand identication system, or ALIS [44]. The ALIS process includes the following steps (see Figure 1): a target receptor plus compound mixture are rst combined in a 96-well plate format and allowed to reach equilibrium, then the plate is loaded into a chromatography system tted with a reusable SEC column containing a proprietary resin for rapid separation (<20 s) of target-ligand complexes from unbound pool components. Proteinligand complexes in the SEC eluant are monitored by UV detection, and an automated valving system directs the protein peak to a reverse-phase chromatography column for dissociation, desalting, and elution of any ligands into an ESIMS system for identication. The ALIS platform has been used to screen a variety of target classes, including integral membrane proteins (Table 3). A collaboration between
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Solution-based AS-MS methods do not require immobilization of the target receptor or ligands on a solid support. This feature avoids the alteration of receptor properties by tagging or chemical linkage, and it allows greater compound diversity since immobilizing functional groups is not necessary. The primary challenge for solutionbased AS-MS methods is the development of efcient techniques to separate receptorligand complexes from unbound small molecules. Separation technologies that use ultraltration membranes are commonly used to isolate high molecular weight biomolecules (>10 kDa proteins and DNA) from small molecules. Researchers at Abbot used ultraltration to develop an AS-MS screening method by (i) combining a library of compounds with a protein, then (ii) separating non-binders by pressure-based ultraltration, (iii) reequilibrating with library-free buffer and re-separating by ultraltration to enrich higher afnity ligands, and (iv) denaturing the retained proteinligand complexes and analyzing the freed ligands by liquid chromatography (LC)MS [32,33]. These researchers identied hits to the important oncology targets Bcl-xL and the mammalian checkpoint kinase CHK1, as well as the anti-infective target Streptococcus enzyme MurF and the oncology target MetAP2 [34,35,36,37]. A disadvantage of ultraltration is that unbound compounds are not completely removed unless subsequent washing steps are employed, which can confound MS detection of true ligands unless they are signicantly enriched relative to chemical noise caused by unbound compounds. In other reports, Sun and van Breemen investigated the relative binding of compounds to estrogen receptors using ultraltration-MSMS [38]. Hannewald et al. reported that centrifugal ultraltration followed by MALDI-MS analysis of the retentate enabled the identication of the control compounds colchicine, vinblastine and vincristine binding to their target receptor tubulin [39]. Cheng and van Breemen also reported an unorthodox, low-throughput AS-MS screening approach where a ligand-induced increase in target solubility, in this case A140 from amyloid precursor protein, was measured by ultraltration-based separation of soluble from aggregated A140 followed by detection of soluble A140 using ow injection ESI-MS [40]. While
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522 Analytical Techniques

Figure 1

Schematic diagram of the ALIS process.

NeoGenesis and Merck yielded a novel inhibitor for the protease -Secretase, an important target for Alzheimers disease therapy [45]. Whitehurst et al. reported on the use of ALIS to screen the muscarinic M2 acetylcholine receptor (M2R), a G-protein coupled receptor (GPCR) [46]. This report was the rst to describe screening an integral membrane receptor by solution-based AS-MS [47]. Beyond HTS, the ALIS platform has been demonstrated to be a valuable analytical tool for characterizing protein ligand interactions [48]. For example, saturation binding assays can be performed where a receptor preparation at constant concentration is titrated with increasing concentrations of a ligand and then analyzed by ALIS to provide absolute binding afnity estimates and to assess
Table 3 Protein types screened by solution-based AS-MS SEC-RPC-ESI-MS Schering-Plough, Merck Kinases Proteases, peptidases Other enzyme classes Nuclear hormone receptors Protein complexes GPCRs, integral membrane proteins Proteinprotein interaction targets Transcription factors U U U U U U U U

the quality of different protein preparations. ALIS enables the simultaneous detection of multiple targetbound ligands in a single binding reaction, allowing the determination of allosteric or isosteric competitive binding proles versus known ligands or cofactors, while also providing mixture-based afnity measurements that enable potency optimization and the development of structureactivity relationships directly from combinatorial chemical libraries [49]. These ALIS-based analytical techniques were highlighted in the report of the ALISbased screening of M2R, where ALIS was applied to three phases of lead discovery: (i) biochemical validation of the target before HTS screening; (ii) HTS screening of a diverse combinatorial small molecule library; and (iii) the characterization of newly discovered hits to determine

GPC Spin column-ESI-MS Amgen, Novartis, Wyeth U U U U

FAC-ESI-MS Protana U U U U

Ultraltration-ESI-MS or -MALDI-TOF-MS Abbott, Van Breemen U U U U

U U

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Affinity selection-mass spectrometry screening techniques for small molecule drug discovery Annis et al. 523

if any are interesting leads [46]. Interestingly, the ALISbased screening of M2R resulted in the discovery of both orthosteric antagonists and a putative allosteric modulator, consistent with the proposed capability of AS-MS to discover ligands with various binding modes for a receptor [50]. To date, Schering-Plough is the only company to have reported the routine use of on-line SEC-LCMS for HTS. However, several proof-of-concept studies have been reported recently that demonstrate other applications of on-line SEC-LCMS. For example, Flarakos and Vouros reported that custom-made SEC columns packed with Sephadex G-25 media (to mimic centrifugal spin-columns) could be connected by an on-line switching system to UV-based detectors, enabling protein ligand complex monitoring and capture for LCMS detection of bound ligandsin this case, compounds that bound to human serum albumin [51]. These investigators reported that improvements in miniaturization are required to reduce protein and compound consumption in their system to enable HTS. Researchers at Merck recently described an integrated SEC-LCMS platform to afnity-rank ligands present in mixtures by competitive afnity selection to dipeptidyl peptidase IV [52]. They showed that the relative afnities of the ligands determined by their AS-MS technique corresponded well with the known relative biochemical IC50 values of the ligands, but did not report on HTS efforts.

quantifying the bound marker after sequential lter isolation and liberation steps. Fragmentation of the marker in a triple quadrupole MS and quantitation of the resulting MSMS ions relative to an isotopically labeled (but non-radioactive) standard yielded excellent reproducibility and good correlation with results from a traditional radioligand analysis [55]. Throughput can also be a challenge for MS-based functional screening. Despite the advent of multi-head injectors for sequential switching between LC-puried samples, MS is an inherently serial analysis technique, since only one sample at-a-time can be introduced to the mass analyzer. (This contrasts with UV- or uorescencebased spectroscopic techniques, where array detectors allow simultaneous measurement from many samples.) To overcome this limitation, researchers at BioTrove have developed an ultra-fast sample switching inlet for a triplequadrupole mass spectrometer, and commercialized its application for monitoring enzyme reaction products by LCMS. Their Rapid Fire system can analyze over 10 000 samples in a day, and was demonstrated by the discovery of several potent inhibitors of acetylcholinesterase, the Akt1 kinase, and the membrane-associated protein phosphatidylserine decarboxylase (PISD) [56,57,58]. In another approach to automated MS-based enzyme assays, Brennan and co-workers have developed a sol gel entrapment system to immobilize proteins for on-line inhibitor screening [59]. By eluting enzyme substrates plus possible inhibitors over the trapped proteins, these researchers measure the effect of the inhibitor on enzyme activity or receptor binding, as exemplied by a screen of 49 compounds against adenosine deaminase (ADA) with IC50 determination for active inhibitors. They have demonstrated the entrapment technique for a variety of target systems, including membrane proteins such as the nicotinic acetylcholine receptor (nAChR) and dopamine D2 receptor [60]. Enzyme inhibitors have also been characterized in highthroughput fashion by MALDI-MS techniques, including desorption/ionization on silicon (DIOS-MS) [61] and from carbon nanotube-based matrices [62]. A particular advantage of the MALDI approach is demonstrated by its application in kinase assays. Traditionally the phosphorylation of a protein or substrate is monitored by incorporation of radioactive phosphate into the product. MALDI-MS allows modication of the native substrate even whole proteins to be monitored directly without the use of a labeled probe [63]. Unfortunately, these approaches are limited to use with volatile buffers, for example, ammonium carbonate.

MS measurements of receptor function

MS-based techniques that monitor functional assays have advantages that parallel those of AS-MS. Target-based functional assays identify lead compounds by measuring the outcome of a biomolecule-mediated transformation, such as production or depletion of the starting materials or products of an enzymatic reaction, or displacement of a labeled marker compound from a biomolecular receptor. These measurements are traditionally done using radiochemical methods, uorescence readouts, or UV spectroscopy. By using MS analysis, the products of enzymatic reactions or displaced ligands can be directly identied by virtue of their molecular mass, which eliminates chemical interferences and obviates the need for radioactive or uorescent labeling of compounds or marker agents. A considerable challenge to MS-based functional screening is that most assays require high concentrations of salts, buffers, cofactors or detergents, which are nonvolatile components that interfere with MS analysis coupling LC with the mass spectrometer enables nonvolatile component removal, and Wanner and co-workers used this capability to develop competitive MS-binding assays [53,54]. In this system, LCMS detection is used to assess the ability of test compounds to compete a native (nonlabeled) marker ligand from its pharmacological receptor, by either analyzing the free (unbound) marker or by
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Conclusions and future directions

As shown in this report, the pharmaceutical industry is enthusiastically using MS-based HTS methods to
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discover and characterize new lead compounds, and we anticipate these applications will continue and expand in the future. Presently, no one-size-ts-all AS-MS solution is expected to satisfy all future screening demands, and no commercial product specically tailored for AS-MS screening is available to interested researchers. Rather, a diversity of methods is implemented by different commercial and academic laboratories, each with its particular advantages and disadvantages, suggesting that no single technique can solve the broad range of requirements that modern pharmaceutical discovery requires. As analytical instrumentation becomes miniaturized and more automated, especially MS and LC components, their ease of use for non-specialists will naturally allow more widespread and creative applications and may expand the already considerable impact of AS-MS on drug discovery.

12. Sinz A: Investigation of Proteinligand Interactions by Mass Spectrometry. Chem Med Chem 2007. 13. Hofstadler SA, Sannes-Lowery KA: Applications of ESI-MS in  drug discovery: interrogation of noncovalent complexes. Nat Rev Drug Discov 2006, 5:585-595. This report provides a comprehensive overview of afnity selection-MS techniques for discovering DNA and RNA ligands. The authors provide examples from their laboratories that demonstrate drug discovery by direct MS detection of noncovalent drug-target complexes. 14. Zhang S, Van Pelt CK: Chip-based nanoelectrospray mass spectrometry for protein characterization. Expert Rev Proteomics 2004, 1:449-468. 15. Zhang S, Van Pelt CK, Wilson DB: Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry. Anal Chem 2003, 75:3010-3018. 16. Xie Y, Zhang J, Yin S, Loo JA: Top-down ESI-ECD-FT-ICR mass spectrometry localizes noncovalent proteinligand binding sites. J Am Chem Soc 2006, 128:14432-14433. 17. Raji MA, Frycak P, Beall M, Sakrout M, Ahn JM, Bao Y, Armstrong DW, Schug KA: Development of an ESI-MS screening method for evaluating binding afnity between integrin fragments and RGD-based peptides. Int J Mass Spectrom 2007, 262:232-240. 18. Clark SM, Konermann L: Determination of ligand-protein dissociation constants by electrospray mass spectrometrybased diffusion measurements. Anal Chem 2004, 76:7077-7083. 19. Clark SM, Konermann L: Screening for noncovalent ligandreceptor interactions by electrospray ionization mass spectrometry-based diffusion measurements. Anal Chem 2004, 76:1257-1263. 20. Makara GM, Athanasopoulos J: Improving success rates for lead generation using afnity binding technologies. Curr Opin Biotechnol 2005, 16:666-673. 21. Cunningham BT, Laing L: Microplate-based, label-free detection of biomolecular interactions: applications in proteomics. Expert Rev Proteomics 2006, 3:271-281. 22. Ng ES, Yang F, Kameyama A, Palcic MM, Hindsgaul O, Schriemer DC: High-throughput screening for enzyme inhibitors using frontal afnity chromatography with liquid chromatography and mass spectrometry. Anal Chem 2005, 77:6125-6133. 23. Chan N, Lewis D, Kelly M, Ng ESM, Schriemer DC: Frontal afnity chromatography mass spectrometry for ligand discovery and characterization. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G. Wiley-VCH; 2007:217246 . [Mannhold R, Kubinyi H, Folkers G (Series Editor): Methods and Principles in Medicinal Chemistry.] 24. Besanger TR, Hodgson RJ, Green JRA, Brennan JD: Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases. Anal Chim Acta 2006, 564:106-115. 25. Besanger TR, Hodgson RJ, Guillon D, Brennan JD: Monolithic membrane-receptor columns: Optimization of column performance for frontal afnity chromatography/mass spectrometry applications. Anal Chim Acta 2006, 561:107-118. 26. Slon-Usakiewicz JJ, Dai JR, Ng W, Foster JE, Deretey E, Toledo-Sherman L, Redden PR, Pasternak A, Reid N: Global kinase screening. Applications of frontal afnity chromatography coupled to mass spectrometry in drug discovery. Anal Chem 2005, 77:1268-1274. 27. Slon-Usakiewicz JJ, Ng W, Dai JR, Pasternak A, Redden PR:  Frontal afnity chromatography with MS detection (FAC-MS) in drug discovery. Drug Discov Today 2005, 10:409-416. This overview of FAC-MS provides an in-depth discussion of its applications and compares the features of this important method with those of other afnity selection-mass spectrometry techniques. 28. Slon-Usakiewicz JJ, Ng W, Foster JE, Dai JR, Deretey E, ToledoSherman L, Redden PR, Pasternak A, Reid N: Frontal afnity www.sciencedirect.com

Acknowledgements
We thank Satish Jindal, Jerry Shipps, Arshad Siddiqui, and our colleagues at SPRI for critical review of this manuscript.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. 2. Korfmacher WA: Principles and applications of LCMS in new drug discovery. Drug Discov Today 2005, 10:1357-1367. Lee MS (Ed): Integrated Strategies for Drug Discovery Using Mass Spectrometry, Hoboken, NJ: Wiley-Interscience; 2005.

3. Wanner K, Hofner G (Eds): Mass Spectrometry in Medicinal  Chemistry, Weinheim: Wiley-VCH; 2007. This timely book includes contributions from several leaders in the eld of afnity selection-mass spectrometry and MS-based drug discovery, plus sections on modern MS instrumentation, principles, and applications in pharmacokinetics. 4. Schermann SM, Simmons DA, Konermann L: Mass spectrometry-based approaches to proteinligand interactions. Expert Rev Proteomics 2005, 2:475-485. Deng G, Sanyal G: Applications of mass spectrometry in early stages of target based drug discovery. J Pharm Biomed Anal 2006, 40:528-538. Geoghegan KF, Kelly MA: Biochemical applications of mass spectrometry in pharmaceutical drug discovery. Mass Spectrom Rev 2005, 24:347-366. Hofstadler SA, Lee M (Eds): International Journal of Mass Spectrometry, Special Issue: Drug Discovery: Elsevier; 2004. Cancilla MT, Erlanson DA: Tethering: fragment-based drug discovery by mass spectrometry. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G. Wiley-VCH; 2007:305-320 . [Mannhold R, Kubinyi H, Folkers G (Series Editor): Methods and Principles in Medicinal Chemistry.]

5.

6.

7. 8.

9. 

Erlanson DA, Hansen SK: Making drugs on proteins: sitedirected ligand discovery for fragment-based lead assembly. Curr Opin Chem Biol 2004, 8:399-406. In this article Erlanson and Hansen describe the state-of-the-art in tethering approaches to drug discovery. 10. Ganem B, Li YT, Henion JD: Observation of noncovalent enzyme-substrate and enzyme-product complexes by ion-spray mass spectrometry. J Am Chem Soc 1991, 113:7818-7819. 11. Ganem B, Henion JD: Going gently into ight: analyzing noncovalent interactions by mass spectrometry. Bioorg Med Chem 2003, 11:311-314.

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chromatography with MS detection of EphB2 tyrosine kinase receptor. 1. Comparison with conventional ELISA. J Med Chem 2004, 47:5094-5100. 29. Gullo VP, McAlpine J, Lam KS, Baker D, Petersen F: Drug discovery from natural products. J Ind Microbiol Biotechnol 2006, 33:523-531. 30. Schou C, Heegaard NH: Recent applications of afnity interactions in capillary electrophoresis. Electrophoresis 2006, 27:44-59. 31. Kovarik P, Hodgson RJ, Covey T, Brook MA, Brennan JD: Capillary-scale frontal afnity chromatography/MALDI tandem mass spectrometry using protein-doped monolithic silica columns. Anal Chem 2005, 77:3340-3350. 32. Comess KM, Schurdak ME: Afnity-based screening techniques for enhancing lead discovery. Curr Opin Drug Discov Devel 2004, 7:411-416. 33. Cloutier TE, Comess KM: Library screening using ultraltration and mass spectrometry. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G. Wiley-VCH; 2007:157184 . [Mannhold R, Kubinyi H, Folkers G (Series Editor): Methods and Principles in Medicinal Chemistry.] 34. Qian J, Voorbach MJ, Huth JR, Coen ML, Zhang H, Ng SC, Comess KM, Petros AM, Rosenberg SH, Warrior U et al.: Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms. Anal Biochem 2004, 328:131-138. 35. Comess KM, Schurdak ME, Voorbach MJ, Coen M, Trumbull JD,  Yang H, Gao L, Tang H, Cheng X, Lerner CG et al.: An ultraefcient afnity-based high-throughout screening process: application to bacterial cell wall biosynthesis enzyme MurF. J Biomol Screen 2006, 11:743-754. In this article, Commess and co-workers describe their GPC-spin column process for rapid screening of compound mixtures to discover inhibitors of the important antibacterial target MurF. 36. Comess KM, Trumbull JD, Park C, Chen Z, Judge RA, Voorbach MJ, Coen M, Gao L, Tang H, Kovar P et al.: Kinase drug discovery by afnity selection/mass spectrometry (ASMS): application to DNA damage checkpoint kinase Chk1. J Biomol Screen 2006, 11:755-764. 37. Kawai M, BaMaung NY, Fidanze SD, Erickson SA, Tedrow JS, Sanders WJ, Vasudevan A, Park C, Hutchins C, Comess KM et al.: Development of sulfonamide compounds as potent methionine aminopeptidase type II inhibitors with antiproliferative properties. Bioorg Med Chem Lett 2006, 16:3574-3577. 38. Sun Y, Gu C, Liu X, Liang W, Yao P, Bolton JL, van Breemen RB: Ultraltration tandem mass spectrometry of estrogens for characterization of structure and afnity for human estrogen receptors. J Am Soc Mass Spectrom 2005, 16:271-279. 39. Hannewald P, Maunit B, Muller JF: Tubulin-binding drug screening by MALDI-TOFMS. Anal Chem 2006, 78:4390-4397. 40. Cheng X, van Breemen RB: Mass spectrometry-based screening for inhibitors of beta-amyloid protein aggregation. Anal Chem 2005, 77:7012-7015. 41. Siegel MM: Drug screening using gel permeation chromatography spin columns coupled with ESI-MS. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G. Wiley-VCH; 2007:65-120 . [Mannhold R, Kubinyi H, Folkers G (Series Editor): Methods and Principles in Medicinal Chemistry.] 42. Mayr LM: Tackling the chemogenomic space by novel screening technologies. Ernst Schering Res Found Worksho 2006:111-173. 43. Brown N, Zehender H, Azzaoui K, Schuffenhauer A, Mayr LM, Jacoby E: A chemoinformatics analysis of hit lists obtained from high-throughput afnity-selection screening. J Biomol Screen 2006, 11:123-130. 44. Annis DA, Athanasopoulos J, Curran PJ, Felsch JS, Kalghatgi K, Lee WH, Nash HM, Orminati J-PA, Rosner KE, Shipps JGW et al.: An afnity selection-mass spectrometry method for the identication of small molecule ligands from self-encoded www.sciencedirect.com

combinatorial libraries: Discovery of a novel antagonist of E. coli dihydrofolate reductase. Int J Mass Spectrom 2004, 238:77-83. 45. Coburn CA, Stachel SJ, Li YM, Rush DM, Steele TG, ChenDodson E, Holloway MK, Xu M, Huang Q, Lai MT et al.: Identication of a small molecule nonpeptide active site betasecretase inhibitor that displays a nontraditional binding mode for aspartyl proteases. J Med Chem 2004, 47:6117-6119. 46. Whitehurst CE, Nazef N, Annis DA, Hou Y, Murphy DM,  Spacciapoli P, Yao Z, Ziebell MR, Cheng CC, Shipps GW Jr et al.: Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by afnity selection-mass spectrometry. J Biomol Screen 2006, 11:194-207. This report describes the rst use of AS-MS for the discovery of agonists and antagonists to a G protein-coupled receptor (GPCR), an important class of targets for modern drug discovery. 47. Whitehurst CE, Annis DA: Afnity selection-mass spectrometry and its emerging application to the high throughput screening of G-protein-coupled receptors. Comb Chem High Throughput Screen, in press. 48. Annis DA, Chuang C-C, Nazef N: ALIS: An afnity selection  mass spectrometry system for the discovery and characterization of proteinligand Interactions. In Mass Spectrometry in Medicinal Chemistry. Edited by Wanner K, Hofner G. Wiley-VCH; 2007:121-184 . [Mannhold R, Kubinyi H, Folkers G (Series Editor): Methods and Principles in Medicinal Chemistry.]. This book chapter provides a denitive overview of ALIS as both a drug discovery engine and a valuable tool for characterizing proteinligand interactions. 49. Annis DA, Nazef N, Chuang CC, Scott MP, Nash HM: A general technique to rank proteinligand binding afnities and determine allosteric versus direct binding site competition in compound mixtures. J Am Chem Soc 2004, 126:15495-15503. 50. Falb D, Jindal S: Chemical genomics: bridging the gap between the proteome and therapeutics. Curr Opin Drug Discov Devel 2002, 5:532-539. 51. Flarakos J, Morand KL, Vouros P: High-throughput solutionbased medicinal library screening against human serum albumin. Anal Chem 2005, 77:1345-1353. 52. Adam GC, Meng J, Athanasopoulos J, Zhang X, Chapman KT: Afnity-based ranking of ligands for DPP-4 from mixtures. Bioorg Med Chem Lett 2007. 53. Zepperitz C, Hofner G, Wanner KT: MS-binding assays: kinetic, saturation, and competitive experiments based on quantitation of bound marker as exemplied by the GABA transporter mGAT1. Chem Med Chem 2006, 1:208-217. 54. Hofner G, Wanner KT: Competitive binding assays made easy with a native marker and mass spectrometric quantication. Angew Chem Int Ed Engl 2003, 42:5235-5237. 55. Niessen KV, Hofner G, Wanner KT: Competitive MS binding assays for dopamine D2 receptors employing spiperone as a native marker. Chembiochem 2005, 6:1769-1775. 56. Ozbal CC, LaMarr WA, Linton JR, Green DF, Katz A, Morrison TB,  Brenan CJ: High throughput screening via mass spectrometry: a case study using acetylcholinesterase. Assay Drug Dev Technol 2004, 2:373-381. Ultra-fast LCMS analysis enables the discovery of novel ligands to this very challenging protein target. 57. Forbes CD, Toth JG, Ozbal CC, LaMarr WA, Pendleton JA, Rocks S, Gedrich RW, Osterman DG, Landro JA, Lumb KJ: Highthroughput mass spectrometry screening for inhibitors of phosphatidylserine decarboxylase. J Biomolec Screen 2007, Published Online May 3, 2007 as doi:10.1177/1087057107301320 58. Quercia AK, Myung J, LaMarr WA, Ozbal CC, Landro JA, Lumb KJ: High-throughput screening by mass spectrometry: comparison with the scintillation proximity assay with a focused le screen of AKT1/PKB. J Biomolec Screen 2007, Published Online May 3, 2007 as doi:10.1177/1087057107300647 59. Hodgson RJ, Besanger TR, Brook MA, Brennan JD: Inhibitor  screening using immobilized enzyme reactor Current Opinion in Chemical Biology 2007, 11:518526

526 Analytical Techniques

chromatography/mass spectrometry. Anal Chem 2005, 77:7512-7519. This innovative, in-line method for monitoring enzyme reactions was used to identify inhibitors for adenosine deaminase and directly determine the Ki values for the inhibitors. 60. Besanger TR, Brennan JD: Entrapment of membrane proteins in solgel derived silica. J SolGel Sci Technol 2006, 40:209-225. 61. Shen Z, Go EP, Gamez A, Apon JV, Fokin V, Greig M, Ventura M, Crowell JE, Blixt O, Paulson JC et al.: A mass spectrometry plate reader: monitoring enzyme activity and inhibition with a

desorption/ionization on silicon (DIOS) platform. Chembiochem 2004, 5:921-927. 62. Hu L, Jiang G, Xu S, Pan C, Zou H: Monitoring enzyme reaction and screening enzyme inhibitor based on MALDI-TOF-MS platform with a matrix of oxidized carbon nanotubes. J Am Soc Mass Spectrom 2006, 17:1616-1619. 63. Greis KD, Zhou S, Burt TM, Carr AN, Dolan E, Easwaran V, Evdokimov A, Kawamoto R, Roesgen J: Davis GF: MALDI-TOF MS as a label-free approach to rapid inhibitor screening. J Am Soc Mass Spectrom 2006, 17:815-822.

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