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Application of mass spectrometry technologies for the discovery of low-molecular weight modulators of enzymes and proteinprotein interactions

Hartmut Zehender1 and Lorenz M Mayr2

In recent years, mass spectrometry has gained widespread use as an assay and screening technology in drug discovery because it enables sensitive, label-free detection of lowmolecular weight modulators of biomolecules as well as sensitive and accurate detection of high-molecular weight modications of biomolecules. Electrospray and matrixassisted laser desorption ionization are the most widely used ionization techniques to identify chemical compounds interfering with enzymatic function, receptorligand binding or molecules modulating a proteinprotein interaction of interest. Mass spectrometry based techniques are no longer restricted to screening in biochemical assay systems but have now become also applicable to imaging of biomolecules and chemical compounds in cell-based assay systems and even in highly complex tissue sections.
Addresses 1 Novartis Institutes for Biomedical Research (NIBR), WSJ-350.1.14, 4002 Basel, Switzerland 2 Novartis Institutes for Biomedical Research (NIBR), WSJ-88.10.19, 4002 Basel, Switzerland Corresponding author: Zehender, Hartmut (

gained widespread application in HTS during the past years because label-free, direct and selective monitoring of an analyte by its molecular mass to charge ratio (m/z), rather than indirect detection of the analytes action, for example by a light-inducing energy transfer event, is less prone to artifacts and straightforward with regards to method development. Difcult-to-measure enzyme reactions, for example are effectively addressed with mass spectrometry (MS) by direct measurement of substrate depletion and/or product accumulation. Similarly, monitoring inhibition of enzymatic reactions via a secondary indicator reaction bears the risk for false positives when using an optical readout, as they may detect compounds that interfere with the indicator reaction rather than the target-based catalysis. Directly monitoring the mass (m/z) of the reaction product from the target enzyme is less prone to artifacts. Especially tandem MS (MS/MS) analysis is benecial for such measurements because of its high specicity and precision in the readout based on mass detection of a unique daughter ion arising from collision-induced fragmentation of the parent compound. Targets with an unknown biological function (orphan targets), or targets with a known function, for which assay development as a functional assay with an optical or radiometric readout would be very tedious or even impossible, can be successfully addressed by MS-based identication of chemical binders to a biological target (afnity selection of binders). This direct detection of binding ligands or the ligandtarget complex by MS has the advantage that neither the ligand nor the target has to be modied by a probe needed for detection, such as a chromophore or a uorophore, routinely used for optical and radiometric readouts. This minimizes the risk for the assay developer to compromise the ligands target afnity or the targets functional integrity. In this review, we want to show the recently published benets of mass spectrometry in drug discovery of small molecules that interfere with disease-related biomolecules. Not only MS-based technologies used for afnity selection and functional high-throughput screening but also the usefulness of MS in secondary hit characterization and lead optimization, as well as in high-resolution imaging of tissue samples, will be described. The high specicity and sensitivity of todays commercially available MS detector types and the ongoing trend to further technological improvements in MS detection are the basis
Current Opinion in Chemical Biology 2007, 11:511517

Current Opinion in Chemical Biology 2007, 11:511517 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.2007.08.031

In pharmaceutical industry, one of the early discovery phase activities is related to high-throughput screening (HTS) of large compound libraries with biomolecular targets that are assumed to be closely linked to a disease phenotype. Both biochemical and cellular HTS formats are used, depending on the targets nature, its embedding in complex signaling pathways and its availability as a puried molecule in sufcient amounts for screening. Today, optical readouts like uorescence or luminescence still predominate for the screening of functionally active targets, such as enzymes or receptors, because of their high sample throughput. Mass spectrometry has

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for the ever increasing use of mass spectrometry in modern drug discovery.

Main review text

Afnity selection MS

Afnity selection technologies were recently reviewed [1,2,3], showing that both homogeneous (i.e. target and compounds are in solution during afnity selection) and heterogeneous (i.e. target or compounds are immobilized on a solid surface) formats are useful [2]. Two similar homogeneous technologies are the SpeedScreen from Novartis [4,5] and the AS-MS technology from NeoGenesis (Schering-Plough) [6,7]. Both technologies use compound pools for screening and separate target-bound from unbound compounds by size exclusion chromatography (SEC) at low temperature. The main difference is the 96-well format used with SpeedScreen for the incubation of target with compounds and the separation of target-bound from unbound compounds. A column array for SEC is used with AS-MS. NeoGenesis uses a time-ofight (TOF) detector with AS-MS and Novartis an ion trap MS with SpeedScreen. The size of the compound pools is 1500 compounds per well with AS-MS and 400 compounds per well with SpeedScreen. More than 50 soluble protein targets from different classes have been screened with SpeedScreen using libraries in excess of 500 000 compounds. SpeedScreen delivers essentially binder information, although orthosteric or allosteric characterization of binders to an enzymatic target is possible by measurement of the displacement of known orthosteric or allosteric binders. AS-MS from NeoGenesis was additionally used for afnity ranking and Kd determinations. In afnity competition experiments, known allosteric or competitive binders of Akt-1 and ZAP-70 kinases were displaced by ligands from a compound library [7]. Binders were characterized by 50% bindertarget complex generation (ACE50), which is similar to the IC50 determinations for enzyme inhibitors. The ACE50 method has also been used to screen membrane fragments with the G-protein-coupled muscarinic M2 receptor (GPCR) and to distinguish between orthosteric and allosteric ligands [8]. Khandekar et al. from GlaxoSmithKline have published a method with covalent modication of the ATP binding site by the ATP analogue 50 -uorosulfonylbenzoyl 50 -adenosine (FSBA), applied to several kinases [6]. FSB labeling was controlled with matrix-assisted laser desorption ionization-time-of-ight (MALDI-TOF) measurement of the labeled peptide fragment after protein digestion with trypsin. For screening, an LC MS method with an electrospray-time-of-ight (ESITOF) instrument was used. The authors suggest their method for secondary validation of selective ATP competitive protein kinase inhibitors.
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Another homogeneous afnity selection-mass spectrometry technology with a TOF-MS detector was disclosed by Abbott [9,10], using ultraltration for the separation of target-bound from unbound compounds. Up to 2700 compounds per mixture were screened, and unbound compounds were eliminated by several cycles of ultraltration. The repeated procedure results in an exponential enrichment of binder molecules so that nally compounds with the highest afnity to the target were the most abundant [9]. The Abbott AS-MS method was successfully applied for HTS with the soluble bacterial enzyme MurF [9] and the kinase Chk1 [10]. Afnity ranking and Kd determinations of binder molecules during medium-to-high-throughput screening have been reported by Protana scientists using frontal afnity chromatography MS (FAC-MS) with a time-ofight MS detector and electrospray ionization (ESI-TOFMS) [11,12]. FAC-MS is a heterogeneous technology, as the target protein is immobilized on solid beads lled into a chromatography column. Potential ligands are continuously infused into the column, and the ligands are detected in the column efuent with different breakthrough volumes, related to their target afnity. Monitoring the m/z values of the ligands by MS delivers sigmoidal front curves, which reect the ligands breakthrough times. Up to 200 compounds were screened per run and up to 10 000 compounds were screened in 24 h [11]. When using a void volume marker with no afnity for the immobilized target and an indicator binder with a known afnity between 0.5 and 50 mM, afnity ranking can be derived from the breakthrough volumes of other binders. Increasing the infused concentrations of both ligand and void marker and monitoring the column efuent lead to staircase curves, from which the breakthrough volumes can be plotted as their reciprocal value against the summed ligand concentrations to obtain the Kd value from the y intercept [11]. FAC-MS was applied to screen proteinprotein interactions (antibodyantigen) [12], receptor domains, kinases and also to differentiate between, for example substrate and ATP binding sites of protein kinase Ca (PKCa) by monitoring the shift of the breakthrough volume when adding a substrate site indicator or an ATP site indicator to the infusate [12]. A less frequently used readout technology in HTS is based on Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Owing to its ultrahigh mass resolution and mass accuracy, this technology can provide information on the chemical composition of ligands, their relative or absolute dissociation constants and their target specicity. This was shown for HTS with RNA as a target by the so-called multi-target afnity/specicity screening (MASS) [13]. Another advantage is the inclusion of natural products extracts into screening, for example from fermentation broths. Natural products extend the lead nding scope to small molecules from microbial or plant

Mass spectrometry in drug discovery Zehender and Mayr 513

origin, which are difcult to address with other MS technologies having lower mass resolution and accuracy. An overview of FTICR in drug discovery is given by Zhang et al. [14].
Detection of modiers of functional target activities

Monitoring the glycosyl transferase catalysis in HTS mode with a range of donor and acceptor substrates using electrospray ionization (ESI) TOF-MS readout and evaluation of enzyme multi-substrate kinetics have been recently described by Yang et al. [15]. Another application of MS readout in functional screening was published by Deng et al. from AstraZeneca [16]. These authors describe an assay to measure the inhibition of the ligase reaction of MurC from E. coli, an enzyme involved in the bacterial peptidoglycan biosynthesis. In an endpoint method with UNAM-Ala-Glu as internal standard, the MurC reaction product alanine-uridine diphosphate-N-acetylmuramic acid (UNAM-Ala) was measured by ion trap MS with electrospray ionization after reversed phase chromatography of the reaction mixture. Linearity of the assay covered a wide UNAMAla concentration range (0.015100 mM). The dynamic range and the assay sensitivity (lower limit of quantitation with LCMS/MS = 0.02 pmol injected onto column) could even be improved with an LCMS/MS method using a triple quadrupole MS detector to monitor the UDP fragment (m/z = 403.1) of UNAM-Ala and UNAMAla-Glu [16]. The Michaelis constant (apparent KM) and maximum reaction velocity (Vmax) for various MurC substrates and for ATP were also determined with this MSbased assay. The authors suggest application of their method for secondary orthogonal screening to conrm hits from a primary HTS. Quantitative monitoring of the enzyme reaction with a range of inhibitor concentrations will provide information about their inhibitory potential (IC50 values). To enhance the sample throughput, multiplexing by means of using four HPLC pumps for a staggered sample injection onto four chromatography columns and monitoring of the sequentially introduced column efuent into the ion source of an MS detector is possible. An alternative is the parallel sample injection onto four HPLC columns and MS analysis with an indexed multiplex sprayer (MUX, Waters Inc.). Both multiplexing approaches are described in the review from Deng et al. [3].
Fragment-based screening

to the target, FBS was claimed to deliver molecules that are likely to have higher ligand efciency than molecules found by conventional screening of molecule libraries [17]. Ligand efciency is dened as the ratio of the free energy of ligand binding to the number of nonhydrogen atoms [17,30]. Higher ligand efciency should translate into more efcient drugs with better pharmacokinetics, lower toxicity and lower molecular mass. Mainly X-ray crystallography, nuclear magnetic resonance (NMR) and also virtual screening are used in FBS, as they provide direct information about binding of small molecules to the target. In some cases, MS has been successfully applied as readout [18]. A fragment-based screening method with MS readout was described by Seth et al. from Isis Pharmaceutical Inc. [19]. The authors found a new class of small molecules, benzimidazoles, that bind to the internal ribosome entry side (IRES) IIa sub domain of RNA from Hepatitis C virus (HCV). Initially, a benzimidazole hit was found with a poor afnity constant (Kd) of 100 mM, but MS-assisted and structure activity relation (SAR) controlled derivatization-afforded benzimidazoles with submicromolar afnity to the target domain.
Detection of covalent target modications

Mass spectrometry has been used to characterize covalent targetligand complexes with site-directed ligand discovery [20]. The so-called Tethering Method (developed by Sunesis Pharmaceuticals) takes advantage of a natural or an engineered protein containing a reactive thiol group in the vicinity of a binding site. A library of small molecules or fragments bearing a disulde side chain is then used for screening. Once such a ligand has bound close to the targets reactive thiol group, a disulde exchange is generated under mild and chemo-selective conditions [21]. The resulting covalent targetligand complex can be characterized by the shift in the targets molecular mass using gentle electrospray ionization-mass spectrometry conditions (ESI-MS) [20].
Inhibitors of proteinprotein interaction

An alternative approach to screening small molecule libraries in lead discovery is fragment-based screening (FBS) with libraries of fragments ranging in mass from 200 to 300 Da. Subsequent assembly of low-afnity fragments can generate hits with higher target afnity than each of the combined fragments and, after optimization, generate with high target afnity [17,18]. As a consequence of combining small fragments with an afnity

Site-directed ligand discovery was used to search for peptides that bind to the general mitochondrial import protein Tom20 [22]. Taking advantage of the single cysteine residue at position 100 in the cytosolic domain of human Tom20, which resides in the periphery of the binding groove for the pre-sequence, Obita et al. tethered diverse mitochondrial aldehyde dehydrogenase presequence motifs by inter-molecular disulde bonds to the cytosolic Tom20 domain via their C-terminal cysteine. Relative afnities of the pre-sequence peptides to the Tom20 domain were investigated by MALDITOF-MS. The generated covalent targetpeptide complex was analyzed with MS to compare the relative mass peak intensities of the pseudo-molecular peptide ions as an indicator for their afnity for the target.
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Protein binding

Specic and non-specic protein binding of potential drug candidates provides an important information during lead optimization, as it has an impact on absorption, distribution, metabolism and excretion (ADME) of the compound. Equilibrium dialysis is widely used for ligandprotein binding and apparently preferred over ultraltration, as it avoids non-specic adsorption of hydrophobic compounds to the lter membrane. Mass spectrometry is an effective means to measure compound binding to a target protein following equilibrium dialysis [23]. Wan et al. from AstraZeneca described such a method for medium-throughput analysis to determine the free fraction of compounds bound to plasma proteins [23]. Pools of up to 10 compounds could be measured in one dialysis well, which enhances the sample throughput compared with conventional single compound analysis. The investigators showed with a variety of different reference compounds that using 10% diluted plasma reveals comparable protein binding data to undiluted plasma, which facilitates assays in case of limited plasma availability. A novel MS-based medium-throughput method to determine afnity constants (Kd) for protein binding of lowmolecular weight compounds was developed at Novartis Pharma by Faller et al. (manuscript in preparation). Their method is based on the experimental setup for permeability determinations by the parallel articial membrane permeability assay (PAMPA) [24]. In Fallers assay, supposed serum albumin binders are added to the wells of a microtitre plate containing buffer. To a second 96-well microtitre plate with wells having a membrane bottom (cut-off of, e.g. 10 kDa), a liquid hexadecane or octanol membrane layer is established before reconstituting the same buffer volume as in the wells containing albumin and compounds. The two plates are superimposed such that the wells containing the liquid membrane of the upper plate dip into the wells of the lower plate. Permeation kinetics of unbound compounds can be obtained from the samples drawn from the upper wells at increasing time points using LCMS analysis with a quadrupole or linear ion trap MS detector. The permeated binder concentrations after incubation with human serum albumin (HSA) are compared with those obtained from the binder without the protein. Afnity constants can be derived from the permeation data with particular software. Although this method was developed to determine compound binding to the main plasma carrier proteins (e.g. HSA, a1 acid glycoprotein), extension to afnity constant determinations for a more general characterization of protein binders is possible and currently assessed in our laboratories.
Compoundtissue interactions

techniques, which allow for spatial localization of a variety of low-molecular weight substances and even high-molecular weight proteins in cells and tissue [25]. It should be noted that the conventionally used technologies such as positron emission tomography (PET), electron microscopy (EM), atomic force microscopy (AFM) and scanning tunnel microscopy (STM) mostly require labeled analytes. In addition, infrared (IR) and Raman spectroscopy as well as NMR technologies have been widely used for imaging but suffer from low spatial and chemical resolution and insufcient detection sensitivity. By contrast, imaging mass spectrometry (IMS) has recently gained a lot of attention as a potent technique since it has demonstrated spatial distribution with even submicron analyte resolution in cells and tissues, ranging from atomic ions to large proteins. With IMS, co-localization of drugs with their pharmacological target can be mapped. Essentially three ionization methods are used in IMS, that is laser desorption ionization (LDI), including matrix-assisted laser desorption ionization (MALDI), ion beam induced desorption (sputtering) for secondary ion mass spectrometry (SIMS) and electrospray ionization. Desorption/ionization in silicon (DIOS) was introduced to avoid interference of the MALDI matrix with the detection of small molecules within the mass range of the matrix forming components [26]. Highest spatial resolution has been reported for SIMS with a lateral resolution of about 50 nm [27]. SIMS was also suggested as being the ideal method for detection of atomic ions and small molecules with a molecular weight <500 Da and also needs the shortest imaging time together with MALDI-IMS [25]. A recent application of MALDI-Qq-TOF-MS for the direct analysis and imaging of the Schering-Plough anti-tumour drug SCH 226374 in intact mouse tumour tissue and compound A in rat brain have been described by Reyzer et al. [27]. Mice were repeatedly dosed with 80 mg/kg SCH 226374 and a tumour section prepared at 12 h post-dose. MALDI-TOF single stage MS (MS1) spectra of the tumour slice, to which sinapinic acid was spotted as a matrix at several discrete areas, did suffer from the too small signal of the M + H protonated drug compared with the overlapping signal from the matrix. By contrast, when using a hybrid MALDI Qq-TOF detector in selected reaction monitoring (SRM) mode, the prominent fragment cluster of the SCH 226374 daughter ion (m/z = 228.1) and its 13C (m/z = 229.1) and 37Cl (m/z = 230.1) isotopes could be spatially located in a tumour tissue section. In a Qq-TOF instrument, the orthogonal time-of-ight reecton replaces the scanning quadrupole of a triple quadrupole MS detector and permits collision-activated dissociation (CAD) of the parent ion (M + H). The resolution of the obtained image depends on the thickness of the bre optic used for laser desorption and was 200 mm in the x-direction and 400 mm in the y-direction. Imaging with MALDI Qq-TOF-MS

Increasing interest in drug discovery towards drug and biomarker monitoring is attributed to imaging
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Mass spectrometry in drug discovery Zehender and Mayr 515

demonstrated the distribution of SCH 226374 over most of the tumour section with a higher enrichment in the outer periphery [27]. With the same technique, the group compared the spatial distribution and intensity of the main fragment (m/z = 225) of discovery compound A in spots on slices from rat brain with its concentration in these spots obtained after tissue homogenization and quantitative LCMS/MS analysis [30]. An internal standard was added to both the matrix solution for MALDI Qq-TOF-MS and to the tissue homogenate for quantitative LCMS/MS analysis, in order to minimize shot variability in MALDIMS imaging and to correct for detection variability in quantitative MS/MS evaluations. The brain tissue data from either method were normalized to the respective maximum response in the different spots and the resulting percentages plotted against the plasma concentration of compound A obtained from the same animal used for the brain determinations. Both responses correlated with R2 = 0.95 for the LCMS/MS and R2 = 0.92 for the MALDI Qq-TOF-MS determinations. This showed that, despite the lack of a quantitative spatial drug location by MALDI Qq-TOF-MS, the drug signal intensity correlates with its concentration. Bunch et al. described sample preparation by pre-coating cellulose membranes with a-cyano-4-hydroxycinnamic acid (CHCA) before tissue blotting to image the skin distribution of the antifungal drug ketoconazole that had been topically applied as a shampoo to porcine skin preparations [28]. For vertical imaging of ketoconazole penetration into skin preparations, a cross-section of the porcine skin was analyzed with the MALDI Q-TOF-MS system. Quantitative estimates of the most abundant sodium adduct of ketoconazole, using the sodium CHCA adduct as an internal standard, showed a good linear response between 0 and 0.4 mg/mL. The investigators succeeded in describing the spatial distribution of the ketoconazoleNa adduct up to 0.8 mm into the skin [28]. Intracellular localization of iodobenzamide in melanoma cells from metastases in mouse lungs was described by Guerquin-Kern et al. using SIMS microscopy [29]. Iodobenzamide is characterized by its high afnity to melanin. A very high lateral resolution 50 nm was achieved with the NanoSims 50 system (Cameca SA, Courbevoie, France) when using caesium primary ions. This system was also used for imaging of iodobenzamide in lung pieces obtained from melanoma tumour bearing mice that had been dosed by an intravenous injection of N-2-diethylaminoethyl-4-iodobenzamide (127I-BZA) 6 h before sacrice. SIMS analyses were done with serial tissue sections using Cs+ as primary ion source. Distribution of CN ions as nitrogen marker for proteins and melanin, P ions as a marker for phosphorus containing nucleic acids and membrane phospholipids and

I (m = 127) ions as the marker for iodobenzamide were recorded as a matrix of 256 256 image points. Lateral resolution was 80100 nm. Melanoma colonies in lung tissue could be distinguished in the slices with light microscopy because of the high melanin content. Phosphorous ions were located by SIMS not only in the cell nucleus, emitted by the nucleic acids, but also in other cellular structures. Melanin-rich grains were free of a phosphorus signal, but their high CN signal perfectly correlated with that coming from I ions of I-BZA or its metabolites. This demonstrated the co-localization of iodobenzamide and melanin and highlighted the imaging potency of SIMS microscopy to study the ultrastructural distribution of a drug within cells [29].

Mass spectrometry is a powerful tool to study interactions of molecules with a wide range of molecular size. Its main advantage over optical or radioactive detection methods is the lack of analyte labeling, the inherent high detection sensitivity and specicity over a large mass range and its versatility in addressing qualitative and/or quantitative analytical aspects. A variety of MS detector types can be selected to cover the different analytical challenges. Detectors and ion sources mentioned in this review, together with their main application, advantages and limitations, are compiled in Table 1. The scope of benecial use of MS detection in HTS ranges from (1) afnity selection methods with orphan or non-tractable targets to an optical functional assay, to (2) functional screening with direct detection of difcult-to-measure enzyme reactants, to (3) screening with low-afnity molecular fragments, to (4) proteinprotein interactions, which would be difcult to address with other technologies, to (5) direct measurement of covalent protein modications and (6) imaging of small molecules in tissue and cells with high spatial resolution, such as drugs or drug-like substances. Not all of these MS-based applications are high throughput, but method development for assays with MS readout is often short, as there is no need to introduce a chromophore or uorophore to any of the analytes. Molecular imaging with MS seems to gain widespread interest as a medium-to-low-throughput secondary assay in support of lead optimization programmes not only in pharmaceutical industry but also in clinical applications to enable tissue-specic drug or biomarker localization. In pharmaceutical industry, the switch from screening with huge libraries of >106 compounds to smaller but focused libraries, which better address particular target classes, for example kinases or proteases, will enhance the use of methods with MS detection. Screening with smaller libraries will compensate for the drawback of time-consuming sequential sample analysis, inherent to mass spectrometry. Although optical detection based assays will continue to cover many of the topics in drug/lead discovery that need high-throughput, labelfree assays with MS detection can be expected to increase
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Table 1 Mass spectrometry detectors and ion sources used in drug discovery Advantages MS type Triple quadrupole (QqQ) High sensitivity; MS/MS capability Limitations Low mass accuracy; restricted mass range (4000 Da) Main application Quantitative measurements with high sensitivity in selected reaction monitoring (SRM) or single ion monitoring (SIM) Monitoring in full scan mode of mass encoded compound pools Discrimination of compounds with mass differences <0.01 Da; measurements of mass shifts by covalent protein modications Whenever extremely high mass accuracy is mandatory Concentration sensitive, quantitative measurements using LC-QqQ-MS, LC-ITMS, or ESI-TOF-MS and ESI-Qq-TOF-MS

Ion trap (IT) (linear, three-dimensional) Time of ight (TOF)

MSn capability; almost no loss in sensitivity when run in full scan mode High mass accuracy and high sensitivity; large mass range >20 000 Da

Low mass accuracy; restricted mass range (4000 Da) Internal standardization often requested

Fourier transform ion cyclotron resonance (FTICR) Ion source Electrospray (ESI)

Extremely high-resolving power; MS/MS capability Soft ionization; miniaturization due to concentration sensitivity (nanoESI); can be used with polar, nonvolatile compounds, for example high MW peptides, proteins, oligonucleotides due to formation of multiply charged ions High mass range (up to 300 kDa); mainly mono-isotopic ions formed; fast and sensitive No interference of small analytes with matrix components used with MALDI Alternative to MALDI; does not need matrix; highest spatial analyte resolution in tissue; shortest imaging time


Ion suppression; mainly used with moderate to highly polar substances; not tolerant to non-volatile salts

Matrix-assisted laser desorption ionization (MALDI) Desorption/ionization in silicon (DIOS) Ion beam induced desorption (sputtering) for secondary ion mass spectrometry (SIMS)

Matrix in the mass range of small analytes; reproducibility Same as MALDI

No widespread use; few commercial equipment sources

MALDI-TOF or MALDITOF/TOF for determinations with high mass accuracy Used instead of MALDI in case matrix components impair analyte detection Used for high-resolution imaging with MS

because the easy-to-address biological targets seem to become scarce, and more difcult targets, requesting more demanding optical or radiation-based detection methods, seem to increase, which can often be better addressed by technologies with MS readout.


Zehender H, Mayr LM: Application of high-throughput afnityselection mass spectrometry for screening of chemical compound libraries in lead discovery. Expert Opin Drug Discov 2007, 2:285-294.


We are very grateful to our colleagues at NIBR (Novartis Institute of Biomedical Research), Center of Proteomic Chemistry (NIBR/CPC) and Department of Discovery Technologies (NIBR/DT), who have contributed with ideas and advice to the implementation and use of MS-based technologies for drug discovery at Novartis, in particular R Amstutz, P Bernet, J Blank, S Cottens, RA Falchetto, B Faller, I Filipuzzi, M Forstner, F Freuler, P Fuerst, F Glickman, M Klumpp, L Leder, F Le Goff, N Lehmann, I Muckenschnabel, J Ottl and C Textor. We fully appreciate their continuous support, encouragement and enthusiasm for novel technologies in drug discovery.

Deng G, Sanyal G: Application of mass spectrometry in early stages of target based drug discovery. J Pharm Biomed Anal 2005, 40:528-538. Good overview on the use of different MS methods in drug discovery. 4. Zehender H, Le Goff F, Lehmann N, Filipuzzi I, Mayr LM: SpeedScreen: the Missing Link between genomics and drug discovery. J Biomol Screen 2004, 9:498-505.

5. Mayr LM, Zehender H: Afnity-based screening. Eur Pharm Rev  2006, 4:30-37. Review of afnity-based high-throughput screening technologies. 6. Khandekar SS, Feng B, Yi T, Chen S, Laping N, Bramson N: A liquid chromatography/mass spectrometry-based method for the selection of ATP competitive kinase inhibitors. J Biomol Screen 2005, 10(5):447-455.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.  Makara GM, Athanasopoulos J: Improving success rates for lead generation using afnity binding technologies. Curr Opin Biotechnol 2005, 16:666-673. Overview on different afnity-based technologies, including also those with MS readout.


Annis DA, Nazef N, Chuang CC, Scott MP, Nash HM: A general technique to rank proteinligand binding afnities and determine allosteric vs. direct binding site competition in compound mixtures. J Am Chem Soc 2004, 126:15495-15503. Interesting description of a novel afnity-based screening technology to determine binding sites and to rank ligand afnities. 8. Whitehurst C, Nazef N, Annis DA, Hou Y, Murphy DM, Spacciapoli P, Yao Z, Ziebell MR, Cheng CC, Shipps GW 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.

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Mass spectrometry in drug discovery Zehender and Mayr 517


Comess K, Schurdak ME, Voorbach MJ, Coen M, Trumbull JD, Yang H, Gao L, Tang H, Cheng X, Lerner CL et al.: An ultraefcient afnity-based high-throughput screening process: application to bacterial cell wall biosynthesis enzyme MurF. J Biomol Screen 2006, 11:743-754. Application of ultra ltration cycles to efciently separate bound from unbound ligands. 10. Comess KM, Trumbull JD, Park Ch, 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. 11. Slon-Usakiewicz JJ, Ng W, Dai J-R, Pasternak A, Redden PR:  Frontal afnity chromatography with MS detection (FAC-MS) in drug discovery. Drug Discov Today 2005, 10(6):409-416. Nice introduction into frontal afnity chromatography mass spectrometry (FAC-MS) technology. 12. Zhu L, Chen L, Luo H, Xu X: Frontal afnity chromatography combined on-line with mass spectrometry: a tool for the binding study of different epidermal growth factor receptor inhibitors. Anal Chem 2003, 75:6388-6393. 13. Sannes-Lowery KA, Cummins LL, Chen S, Drader JJ,  Hofstadler SA: High throughput drug discovery with ESI-FTICR. Int J Mass Spectrom 2004, 238:197-206. Nice presentation of an FTICR method to target regions of RNA with small molecules using multi-target afnity/specic screening (MASS). 14. Zhang J, McCombie G, Guenat C, Knochenmuss R: FT-ICR mass  spectrometry in the drug discovery process. Drug Discov Today 2005, 10(9):635-642. Good introduction in principle and some applications of FTICR mass spectrometry in drug discovery. 15. Yang M, Brazier M, Edwards R, Davis BG: High-throughput  mass-spectrometry monitoring for multisubstrate enzymes: determining the kinetic parameters and catalytic activities of glycosyltransferases. Chem Biochem 2005, 6:346-357. Nice description of the use of MS for determination of kinetic parameters of multi-substrate enzymes. 16. Deng G, Gu R-F, Marmor S, Fisher SL, Jaric H, Sanyal G: Development of an LCMS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis. J Pharm Biomed Anal 2004, 35:817-828. 17. Erlanson DA: Fragment-based lead discovery: a chemical  update. Curr Opin Biotechnol 2006, 17:643-652. Interesting presentation and comparison of technologies used in fragment-based screening. 18. Jahnke W, Erlanson DA: Fragment-based approaches in drug discovery. In Methods and Principles in Medicinal Chemistry (34). Edited by Mannhold R, Kubinyi H, Folkers G. Weinheim, Germany: Wiley-VCH; 2006. 19. Seth PP, Miyaji A, Jefferson EA, Sannes-Lowery KA, Osgood SA,  Propp SS, Ranken R, Massire C, Sampath R, Ecker DJ et al.: SAR

by MS: discovery of a new class of RNA-binding small molecules for the Hepatitis C virus: internal ribosome entry Site IIA subdomain. J Med Chem 2005, 48:7099-7102. Discovery of new small molecules that bind to the IRES subdomain of hepatitis C virus RNA using MS-based screening. 20. Erlanson DA, Hansen SK: Making drugs on proteins: site directed ligand discovery fragment based lead assembly. Curr Opin Chem Biol 2004, 8:399-406. 21. Erlanson DA, Wells JA, Braisted AC: Tethering: fragment based drug discovery. Annu Rev Biophys Biomol Struct 2004, 33:199-223. Exhaustive description of the fragment-based screening technology tethering. 22. Obita T, Muto T, Endo T, Kohda D: Peptide library approach with a disulde tether to rene the Tom20 recognition motif in mitochondrial presequences. J Mol Biol 2003, 328:495-504. 23. Wan H, Rehngren M: High-throughput screening of protein binding by equilibrium dialysis combined with liquid chromatography and mass spectrometry. J Chromatogr A 2006, 1102:125-134. 24. Kansy M, Senner K, Gubenator K: Physicochemical high  throughput screening: parallel articial membrane permeation assay in the description of passive absorption processes. J Med Chem 1998, 41(7):1007-1010. Introduction into PAMPA technology. 25. Rubakhin SS, Jurchen JC, Monroe EB, Sweedler JV: Imaging  mass spectrometry: fundamentals and applications to drug discovery. Drug Discov Today 2005, 10(12):823-836. Nice overview on imaging using MS in drug discovery. 26. Wei J, Buriak JM, Siuzdak G: Desorption-ionization mass spectrometry on porous silicon. Nature 1999, 399:243-246. 27. Reyzer ML, Hsieh Y, Ng K, Korfmacher WA, Caprioli RM: Direct analysis of drug candidates in tissue by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom 2003, 38:1081-1092. 28. Bunch J, Clench MR, Richards DS: Determination of pharmaceutical compounds in skin by imaging matrixassisted laser desorption/ionisation mass spectrometry. Rapid Commun Mass Spectrom 2004, 18(24):3051-3060. 29. Guerquin-Kern JL, Hillion F, Madelmont JC, Labarre P, Papon J,  Croisy A: Ultra-structural cell distribution of the melanoma marker iodobenzamide: improved potentiality of SIMS imaging in life sciences. Biomed Eng Online 2004, 3:10-16. Interesting presentation of secondary ion mass spectrometry (SIMS) to image a melanoma marker in tissue with high resolution. 30. Hopkins AL, Groom CR, Alex A: Ligand efciency: a useful metric for lead selection. Drug Discov Today 2004, 9(10): 430-431.

Current Opinion in Chemical Biology 2007, 11:511517