Kinetic studies of small molecule interactions with protein kinases using biosensor technology

ANALYTICAL

BIOCHEMISTRY

Analytical Biochemistry 340 (2005) 359–368

www.elsevier.com/locate/yabio

Kinetic studies of small molecule interactions with proteinkinases using biosensor technology

Helena Nordin, Maria Jungnelius, Robert Karlsson, Olof P. Karlsson *

Department of Biochemistry and Chemistry, Biacore AB, Rapsgatan 7, SE-754 50 Uppsala, Sweden

Received 20 December 2004Available online 11 March 2005

Abstract

Protein kinases are among the most commonly targeted groups of molecules in drug discovery today. Despite this, there are fewexamples of using surface plasmon resonance (SPR) for kinase inhibitor interaction studies, probably reflecting the need for betterdeveloped assays for these proteins. In this article, we present a general methodology that uses biosensor technology to study smallmolecule binding to eight different serine/threonine and tyrosine kinases. Mild immobilization conditions and a carefully composedassay buffer were identified as key success factors. The methodology package consists of direct binding studies of compounds toimmobilized kinases, kinase activity assays to confirm inhibitory effects, detailed kinetic analyses of inhibitor binding, and compe-tition assays with ATP for identification of competitive inhibitors. The kinetic assays resolve affinity into the rates of inhibitor bind-ing and dissociation. Therefore, more detailed information on the relation between inhibitor structure and function is obtained. Thismight be of key importance for the development of effective kinase inhibitors.� 2005 Elsevier Inc. All rights reserved.

Keywords: Surface plasmon resonance; Protein kinase inhibitors; Drug discovery; Methodology; Binding rate constants; Biosensor

1 Abbreviations used: SPR, surface plasmon resonance; GST, gluta-thione S-transferase; c-Src, cellular sarcoma phosphoprotein; ERK2,extracellular signal-regulated kinase 2; CDK2, cyclin-dependent kinase

Kinases are a large family of proteins that catalyzethe transfer of the c-phosphate group of ATP or GTPto the hydroxyl group of serine, threonine, or tyrosineon a substrate protein, often as a response to membranereceptor activation. Cellular signaling cascades rely onthe phosphorylation status of proteins in their path-ways. As many as one-third of all human proteins arethought to be substrates for protein kinases [1,2]. Mal-functions in these processes can have pathological con-sequences such as cancer and inflammatory disease.This makes protein kinases one of the most importanttarget groups for drug discovery [3,4]. The largest groupof kinase inhibitors is the ATP competitors [2].Although many high-affinity compounds have beendeveloped, specificity is often a problem because mostkinases have a highly conserved ATP binding site [2].

0003-2697/$ - see front matter � 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2005.02.027

* Corresponding author. Fax: +46 18 150113.E-mail address: [email protected] (O.P. Karlsson).

Biacore�s surface plasmon resonance (SPR)-based1

protein interaction technology provides information-rich data on biomolecular interactions and has beenrecognized as a powerful tool for drug discovery appli-cations [5]. SPR biosensors enable the interaction be-tween biomolecules to be monitored in real time usinglabel-free assay formats. One of the interacting mole-cules, the ligand, is immobilized on a biosensor surface.The other molecule, the analyte, is then injected in themobile phase in contact with the surface. As analytesfrom the injected sample bind to the ligand attached

2; CamK4, calmodulin-dependent kinase 4; DMSO, dimethyl sulfox-ide; MBP, myelin basic protein; DTT, dithiothreitol; SAR, structure–activity relationship; QSAR, quantitative structure–activity relation-ship; CSK, c-src kinase.

mailto:[email protected]

360 Interactions with protein kinases using biosensor technology / H. Nordin et al. / Anal. Biochem. 340 (2005) 359–368

to the surface, a change in SPR response is detected. Thechange in response level after the injection is propor-tional to the change in mass at the surface.

Binding curves corresponding to complex formationand dissociation are recorded in real time. From theshape of the binding curves kinetic rate constants i.e.,association rate (ka) and dissociation rate constants(kd), can be derived by fitting the data to appropriateinteraction models [6]. In drug discovery lead series,the kinetic rate constants can vary by several orders ofmagnitude even when compounds have similar affinitiesgiven that KD = kd/ka [7–9]. This means that determina-tion of affinities alone does not provide an adequate ba-sis for lead selection.

There are very few examples in the literature ofanalyzing small molecule interactions with kinasesusing SPR biosensors, and those studies mainly con-cern the mitogen-activated protein kinase p38[10,11]. Here we present a methodology that was ap-plied successfully to studies of eight different kinases,representative of this large protein family. Immobili-zation conditions were found to be critical, and sev-eral strategies for preserving the binding capacityfor inhibitors and ATP were developed. The composi-tion of the assay buffer was developed for optimizedkinase assay performance. The assays presented in-clude kinetic characterization of inhibitor binding tokinases and analysis of binding characteristics in thepresence of ATP for identification of ATP-competitivebinders. The effect of kinase phosphorylation on thebinding of ATP and inhibitors was also investigated,as was the kinase isotype specificity. Furthermore, acomplementary activity assay was used to investigatewhether there was a correlation between binding tokinase and enzyme inhibition. All of this togethersuggests that the biosensor methodology that we havedeveloped offers a useful tool in kinase-targeted drugdiscovery.

Materials and methods

Materials

Biacore S51 and Biacore 3000, Sensor Chip CM5(classic and Series S), amine coupling kit, HBS-P buffer,glutathione S-transferase (GST), and anti-GST were ob-tained from Biacore. Biacore instruments are based onSPR detection at gold surfaces and a microfluidic systemfor reagent handling.

All p38 kinases, cellular sarcoma phosphoprotein (c-Src), extracellular signal-regulated kinase 2 (ERK2), cal-modulin (bovine brain), and inhibitors SB 203580 and202190 were obtained from Upstate Biotechnology.Inhibitors PD 169316, 5-iodotubercidin, hypericin, andSU 6656 were purchased from Calbiochem. PP1 and

radicicol were purchased from Biomol International.Staurosporin was obtained from Sigma. Anti-penta-His antibody was obtained from Qiagen, and anti-biotinantibody, clone B-E24, was obtained from Diaclone. Ki-nase inhibitors A, 1A to 1F, Schering kinase 2, Scheringkinase 3, and cyclin-dependent kinase 2 (CDK2) weregifts from Schering. Kinases c-Src kinase (CSK) and cal-modulin-dependent kinase 4 (CamK4) were gifts fromProtometrix.

Immobilization of capture antibodies

Amine coupling of anti-GST, anti-penta-His, andanti-biotin antibodies on Sensor Chip CM5 was per-formed according to the surface preparation wizard inthe Biacore instrument control software, including thefollowing steps: (i) activation with 0.2 M N-ethyl-N-dimethylaminopropylcarbodiimide and 50 mM N-hy-droxysuccinimide for 7–10 min; (ii) injection for15 min (5 ll/min) in 5 mM maleate (pH 6.0) for anti-GST, injection for 10 min (10 ll/min) in 10 mM acetate(pH 5.0) for anti-penta-His antibody, and injection for10 min in 10 mM acetate (pH 5.5) for anti-biotin anti-body, with all three antibodies at 30 lg/ml; and (iii) sur-face blockage with 1.0 M ethanolamine (pH 8.5) for7 min.

Immobilization of kinases

Kinases (25–30 lg/ml) were amine coupled underconditions specified in Table 1 according to the sameprocedure as for antibodies described above.

Capture was performed by injecting the GST or Hiskinase fusion protein over amine-coupled anti-penta-His or anti-GST antibodies at concentrations of 3–30 lg/ml in buffer (Table 1) for 10–15 min at a flow rateof 10 ll/min using a custom method for surface prepara-tion in the instrument control software. Injections wererepeated until saturation or until the desired level wasreached. The GST capture resulted in a stable baseline,whereas His-captured kinase was stabilized by surfaceactivation with 0.2 M N-ethyl-N-dimethylaminopropyl-carbodiimide and 50 mM N-hydroxysuccinimide for2.5 min and blocking with 1.0 M ethanolamine (pH8.5) for 2.5 min (cross-linking).

Binding experiments

Compounds were stored as 10 mM stock solutions in100% dimethyl sulfoxide (DMSO). All light-sensitivecompounds were handled under yellow protective light.After dilution to 1 mM with 100% DMSO, samples weremixed with 1.03- to 1.05-fold concentrated assay bufferand DMSO to yield 10 lM compound in a final buffercomposition of 50 mM Tris (pH 7.5), 150 mM NaCl,10 mM MgCl2, 1 mM MnCl2, and 3 or 5% DMSO

Table 1Use of four different kinase immobilization strategies to immobilize each of eight kinases

Kinase Immobilizationa Coupling buffer Typical level (RU) Binding capacityb (%)

CDK2 Amine coupled 10 mM sodium borate (pH 7.75) 4500 40Schering 2 Amine coupled 100 mM sodium borate (pH 7.75), 5 mM DTT 7000 70ERK2 Amine coupled protected 10 mM sodium acetate (pH 5.5), 2 mM MgCl2, 1 mM ATP 9200 60c-Src Amine coupled protected 10 mM sodium borate (pH 7.0), 2 mM MgCl2, 1 mM ATP 8000 40p38a Amine coupled protected 10 mM sodium acetate (pH 5.5), 10 lM SB 203580 11000 25p38a/p38c GST capture PBS 2200 90CSK GST capture PBS 2300 40CamK4 GST capture PBS 1600 25Schering 3 His capture + cross-link Assay buffer 4000 50

Note. RU, resonance units; CDK2, cyclin-dependent kinase 2; ERK2, extracellular signal-regulated kinase 2; c-Src, cellular sarcoma phosphoprotein;PBS, phosphate-buffered saline; CSK, c-Src kinase; and CamK4, calmodulin-dependent kinase 4.a The specific conditions found for each kinase were optimized and applied using the methodology described in Materials and methods.b Binding capacity is defined as the experimentally determined maximal response in relation to the maximal response stoichiometrically calculated

from the immobilization level of kinase.

Interactions with protein kinases using biosensor technology / H. Nordin et al. / Anal. Biochem. 340 (2005) 359–368 361

(assay buffer), which was also used as the instrumentrunning buffer and sample dilution buffer. ATP was dis-solved directly in running buffer to make a 10 mM solu-tion. The run was started with five start-up cycles whererunning buffer was injected instead of sample, followedby sample injection cycles. Zero concentration sampleswere used as blanks. A typical analysis cycle consistedof a 60 s sample injection (30 ll/min), 120–200 s of buf-fer flow (dissociation phase), followed by a needle andtubing wash with 50% DMSO and buffer, and finally a30 s buffer injection to check for sample carryover.The flow cell temperature was 25 �C. Between sampleseries, a solvent correction cycle was run according tothe instrument manual [12] to adjust for referencing er-rors due to refractive index mismatches between runningbuffer and samples.

Biacore S51 evaluation software (version 1.2.1) wasused for curve fitting of the entire inhibitor concentra-tion series data set (global fit) using numerical calcula-tions of the equation system

A½0� ¼ 0

dA=dt ¼ kt � ðConc� AÞ � ðka � A � B� kd � ABÞ;

where A is the concentration of analyte at the sensor sur-face, B is the immobilized interactant, and Conc is theinjected analyte concentration.

B½0� ¼ Rmax

dB=dt ¼ �ðka � A � B� kd � ABÞ

AB½0� ¼ 0

dAB=dt ¼ ðka � A � B� kd � ABÞ

Total response

ABþRI;

where RI is the refractive index mismatch between thesample and the running buffer.

Kinase enzymatic activity measurements

The kinase (10 lg/ml), biotinylated myelin basic pro-tein (50 lg/ml, MBP), ATP (100 lM), and the inhibitor,at 50 ll final volume, were incubated at 30 �C for 10 minin assay buffer. The reaction was stopped by putting thetube into boiling water for 30 s. The reaction mixturewas injected for 3 min over an immobilized anti-biotinantibody surface, and the potentially phosphorylatedMBP was captured. The phosphorylation status of thekinase substrate was detected by a 3-min injection(5 ll/min) of 10 lg/ml anti-phospho-MBP. The differ-ence in response before and immediately after the injec-tion was used as the enzyme activity value. Theimmobilized capture antibody was regenerated beforethe next sample with 0.1% trifluoroacetic acid for 30 sat 10 ll/min. HBS-P was used as running buffer.

For inhibition analysis, the data from an inhibitorconcentration interval were fitted, assuming a 1:1 stoi-chiometry, to the equation

Response ¼ Responsehigh � fðResponsehigh �ResponselowÞ=½1þ ðConcentrationinhibitor=IC50Þn�g:

No significant differences in the determined IC50 valueswere observed if n was set to 1 or was fitted as a param-eter, resulting in n values of 0.7–1.1.

Results

Immobilization

The preservation of kinase binding capacity for ATPand inhibitors on immobilization was identified as oneof the most important aspects during the developmentof the methodology for SPR biosensor studies. Four dif-ferent main immobilization strategies were employedduring this study of the eight kinases (Table 1): (i) cova-


lent amine coupling at pH > 5.5, (ii) covalent amine cou-pling with a protective specific binder, (iii) capture withantibody, and (iv) capture with antibody followed bycovalent amine cross-linking. Electrostatic preconcen-tration to the anionic carboxy dextran surface is nor-mally carried out at low ionic strength and pH belowthe isoelectric point during covalent coupling of proteins[13]. This presents an obstacle for some kinases that arenotoriously sensitive to pH < 5.5, losing their bindingcapacity, although this was not the case for all of the ki-nases tested. CDK2 and Schering kinase 2 had relativelyhigh isoelectric points and both preconcentrated at pH7.75, whereas c-Src preconcentrated at pH 7.0. Thisdemonstrates the importance of performing preconcen-tration scouting over a sufficiently wide pH range.

For p38a, useful preconcentration was not possiblewith coupling buffers of pH > 5.5, whereas the capacityof the immobilized enzyme to bind ATP and inhibitorswas irreversibly eliminated at pH 5.5. If, however,10 lM of the inhibitor SB 203580 was included in thecoupling buffer, 25% of the calculated binding capacitycould be retained. Similar observations were made byCasper et al. [11], who also found a thermal destabiliza-tion of the kinase under low-pH conditions. In ourhands, subjecting an immobilized p38a surface of knownbinding capacity to a 30 min injection of coupling bufferresulted in a total extinction of SB 203580 binding. Thesame treatment in the presence of 10 lM SB 203580 pre-served just under 50% of the capacity (Fig. 1). This sug-gests that it is the low pH, and not the coupling

Fig. 1. pH sensitivity of p38a. By duplicate injections of 0.25 lM SB203580, the binding capacity of immobilized p38a was examined aftera 30 min exposure to running buffer (50 mM Tris (pH 7.5), 150 mMNaCl, 10 mM MgCl2, 1 mM MnCl2, and 5% DMSO), coupling buffer(10 mM sodium acetate, pH 5.5), or coupling buffer with 10 lM of theinhibitor SB 203580. The binding capacity of the kinase was eliminatedby the exposure to coupling buffer but was partly protected by thepresence of inhibitor. RU, resonance units.

procedure itself, that is harmful to the kinase and thatthe inhibitor had only a partially protective effect. Whenthe same p38a preparation was immobilized by antibodycapture of its GST tag, 90% of the binding capacity waspreserved, emphasizing that immobilization per se is notharmful to the enzyme. ERK2 and c-Src retained bindingcapacity when amine coupled in the presence of 1 mMATP at pH 5.5 and pH 7.0, respectively (Table 1).

Capture of GST fusion proteins by an immobilizedpolyclonal anti-GST antibody did not yield as highlevels of kinase on the sensor surface as did amine cou-pling. On the other hand, the fraction of enzyme activelybinding analytes was generally sufficiently high, proba-bly because the buffer conditions used during capturecould be adapted to the enzyme. This and the high-sta-bility capture was the major advantage of this approach.Capture of His-tagged Schering kinase 3 did not resultin a stable baseline; however, this was overcome byincluding a covalent amine cross-linking step after thecapture injection.

Buffer optimization

A sample dilution and running buffer used by kinasesuppliers for kinase enzyme activity measurements (e.g.,50 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM MnCl2,1 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 0.001%NP40, and 3% DMSO) was found to be unsuitable forthe Biacore experiments. Although binding of inhibitorsand ATP to the kinases was evident using this buffer,there was also significant unwanted binding to the refer-ence surface. Because the degree of background bindingmight differ between the reference and the kinase detec-tion spots, it cannot be correctly compensated for by ref-erence subtraction. Variable and drifting baselinesobserved with this buffer also complicated reliable ki-netic evaluations of the data. The reference bindingbehavior was easily abolished by adding 0.15 M NaClto the buffer, indicating an electrostatic nature of theinteraction that could be quenched by increased ionicstrength. The baseline drifts were diminished by simpli-fying the buffer composition, taking out all componentsthat did not prove to be necessary for the Biacore bind-ing assay, and by replacing Hepes as the buffer compo-nent. Phosphate- and Tris-based buffers both resulted instable baselines. However, phosphate precipitates withmanganese, which was found to be an essential cofactorfor ATP binding by Schering kinase 3. Because manga-nese [14] and magnesium [15] dependence has been re-ported for other kinases, these salts were included inthe buffer. Consequently, Tris was identified as the pre-ferred buffering agent. The final buffer composition was50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 10 mMMgCl2, 1 mM MnCl2, and 3–5% (v/v) DMSO (Fig. 2).This buffer was used successfully for all of the kinaseexperiments in this study. The DMSO concentration

Fig. 2. Importance of buffer composition. Concentration series (0.01–2 lM) of inhibitor A were run over a Schering kinase 3 surface using twodifferent running and sample buffers: 50 mM Hepes (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 1 mM MnCl2, and 3% DMSO (left panel) and 50 mMTris (pH 7.5), 150 mMNaCl, 10 mMMgCl2, 1 mMMnCl2, and 3% DMSO (right panel). A more stable baseline was achieved when using Tris-basedbuffer. RU, resonance units.

Fig. 3. Screening for binders. A total of 22 different compounds withknown binding specificities for various protein kinases were used forscreening. p38a-GST was immobilized by capture to an anti-GSTantibody and was addressed with the compounds at a concentration of10 lM. The association and dissociation responses for each samplewere read off as marked in the inset sensorgram. The responses wereadjusted for the molecular weight of each compound. The data pointsfor the three p38a binders were clearly separated from the others. RU,resonance units.


was adapted to the solubility of the binders rather thanto the requirements of the enzyme.

Screening for binders

Initially, 22 kinase inhibitors with previously re-ported specificities were tested for their binding top38a by using a screening variant of the direct bindingassay. In screening, only a single concentration of eachcompound was injected, and instead of evaluating theentire sensorgram, two baseline-subtracted data pointswere used: one from the association phase and the otherfrom the dissociation phase. The responses were ad-justed for the molecular weight of each compound sothat compounds could be compared in a plot reflectingthe extent of binding and binding stability. Data forthe three compounds known to be p38a binders weredistinctly separated from data for the others (Fig. 3).Hence, this screening approach proved to be a usefultool for the rapid identification of specific binders.

Inhibition assay and IC50 determinations

To test whether specific kinase binders are enzymeinhibitors, the kinase enzyme activity assay was run inthe presence of binders, as described in Materials andmethods. Variation of binder concentrations aboveand below the affinity equilibrium constant (KD) al-lowed determination of IC50 values for the variousinhibitors (Fig. 4). The IC50 values were generally higherthan the KD value, and this was expected given that theinhibitor competes with ATP for the same binding site inthe activity assay.

Kinetic characterization

The interaction of small molecule inhibitors and ATPwith immobilized kinase could be analyzed for most of

the kinases in the study, providing kinetic and/or affinitydata (Table 2). For CDK2 that must be activated by cy-clin to bind ATP, no ATP binding could be measured.For CamK4, no small molecule inhibitors were avail-able, but data on calmodulin binding and ATP affinitywere obtained. The majority of the inhibitor–kinaseinteractions showed binding kinetics suitable for deter-mination of the kinetic rate constants (Table 2 andFig. 5), whereas the ATP binding to all kinases testedshowed fast on-and-off rates outside the range for quan-titative evaluation. In these cases, the KD value was

Fig. 4. Determination of IC50 values. The activity assay was run, asdescribed in Materials and methods, for p38a in the presence of theATP-competitive inhibitors, yielding IC50 values of 180 nM for SB203580, 120 nM for SB 202190, 90 nM for SB 220025, and 130 nM forPD 169316. The plot shows the data points and the fitted curves fromthe IC50 determinations.


calculated from equilibrium responses of the ATP injec-tions at various concentrations. Plots of the logarithmsof the rate constants against each other were used forcomparing the binding kinetics of different compoundswith the same kinase. For both p38a and CDK2, thevariations in affinities (KD) among binders were due tovariations close to one order of magnitude in both thedissociation rate constant (kd) and the association rateconstant (ka), which would have been obscured by usingonly KD or IC50 values for comparison (Fig. 5). The ben-efit of the evaluation support that this kind of plot cangive increases with the number of data points. Forexample, when plotting large numbers of data, clustersof points can be identified, facilitating classification ofbinders [16].

Specificity assay

The flow cell configuration in Biacore S51 wasused for testing the specificity of compounds, with

Table 2Measurements performed with different kinases

Kinase Type Measu

CDK2 Ser/Thr SmallERK2 Ser/Thr Smallc-Src Tyr ATP ap38a/p38c Ser/Thr ATP aCSK Tyr ATP aCamK4 Ser/Thr CalmoSchering 2 Not stated ATP aSchering 3 Not stated ATP a

Note. CDK2, cyclin-dependent kinase 2; ERK2, extracellular signal-regulateand CamK4, calmodulin-dependent kinase 4.a The specific measurements for each kinase were performed as generally d

p38a immobilized on one detection spot and p38cimmobilized on the other. When injecting a concen-tration series of inhibitor SB 203580, binding was de-tected only in the spot with p38a (Fig. 6). Inaddition, no unwanted binding to the capturing mol-ecule (anti-GST pAb) or to the GST fusion proteinwas detected.

Binding site specificity

Sensorgrams from binding of mixtures of ATP and asimilar sized inhibitor to Schering kinase 3 were com-pared with sensorgrams from ATP and inhibitor bind-ing alone. The comparison showed that increasing theconcentration of ATP transformed the slow inhibitorkinetics into fast ATP-like kinetics (Fig. 7) without add-ing to the total response level.

This demonstrates that the two binders compete forthe binding because the shape of sensorgrams reflectsthe binder that occupies the binding site. For noncom-petitive binders, the sensorgrams would have an inter-mediate appearance, corresponding to the sum of thetwo binding events. This assay setup can also be usedto find out whether inhibitors with different kineticappearances compete for binding, thereby mappingbinding competition among a set of different binders,including ATP.

Binding to active and inactive kinase

A binding assay with phosphorylated (active) andunphosphorylated (inactive) p38a in each of the detec-tion spots showed a strong preference for ATP bindingto the activated form, whereas the inhibitor boundequally to both forms quantitatively as well as qualita-tively (Fig. 8). This revealed a discrepancy betweeninhibitor binding and ATP binding, even though theinhibitor is ATP competitive. Competition experimentswith p38a, such as the one presented for Schering kinase3 (Fig. 7), revealed competitions between ATP and theinhibitors SB 203580 and SB 202190 for active p38a,

rementsa

molecule binding kineticsmolecule binding affinity, enzyme activity/inhibitionffinity and small molecule binding kinetics, enzyme activityffinity and small molecule binding kinetics, enzyme activity/inhibitionffinity and small molecule binding kineticsdulin binding, ATP affinityffinity and small molecule binding kineticsffinity and small molecule binding kinetics

d kinase 2; c-Src, cellular sarcoma phosphoprotein; CSK, c-Src kinase;

escribed in Materials and methods.

Fig. 5. Kinetic characterization of inhibitor binding to CDK2 and p38a. To determine the affinities and kinetic rate constants, inhibitorconcentrations above and below the KD value (typically 4–6300 and 10–500 nM, respectively) were injected over immobilized CDK2 and p38a,respectively. ATP (3–200 lM) and inhibitor 1 A had association and dissociation rates outside the range of the methodology; KD values could becalculated from the association equilibrium data. Sensorgrams (in color) and curve fit (black lines) (assuming 1:1 interactions) are shown in the leftpanels. In the right panels, the ka (recognition) is plotted against the kd (complex stability) on logarithmic scales. RU, resonance units.

Fig. 6. Kinase isoform specificity of inhibitor binding. The parallel detection spots in Biacore S51 were used for simultaneous analysis of SB 203580(10–250 nM) binding to p38a (left panel) and p38c (right panel). RU, resonance units.


Fig. 7. Detection of binding site competition. Comparison of ATP andinhibitor binding alone with binding from mixtures revealed that whenthe concentration of ATP was increased (0–50 lM), the binding andthe slow dissociation characteristic of the inhibitor (0.125 lM) wasgradually extinguished and binding curves became more similar tothose of ATP alone. RU, resonance units.

Fig. 8. Influence of kinase activation on binding properties. The parallel det203580 (10–250 nM) (upper panels) and ATP (3–100 lM) binding (lower pan(inactive) p38a (right panels). RU, resonance units.


in contrast to inactive p38a, where ATP could not com-pete with the inhibitors. These observations are in agree-ment with results from enzyme activity measurementsand radioligand binding experiments [17] as well asstructural data showing that the inhibitors bind to theATP pocket [18].

Discussion

General applicability of the kinase assay conditions

The relatively large number of kinases included inthis study (Table 2) made it possible to identify twoimportant success factors during assay development.The first one was to immobilize sufficient levels of the ki-nase while maintaining its binding capacity. As shown inTable 1, at least 40% of the calculated binding capacitywas retained for seven of nine kinases examined (range40–90%). The sensitivity of the method was demon-strated in the case of CamK4. Here, despite a low immo-bilization level (1600 RU) and significant loss of ATPbinding capacity (only 25% retained), useful data were

ection spots in Biacore S51 were used for simultaneous analysis of SBels) to phosphorylated (active) p38a (left panels) and unphosphorylated


still achieved (2.5 RU maximum response). A key factorin maximizing binding capacity was to avoid pH levelsbelow 6.0 during kinase immobilization. For p38a andCDK2, for example, it was demonstrated that treatmentof immobilized and active kinase with low pH bufferabolished all of the binding capacity for inhibitor.P38a could, however, be partially protected by the pres-ence of inhibitor in the low-pH buffer, which was usedwhen electrostatic preconcentration was required duringimmobilization (Fig. 1). The other kinases in the studyeither could be covalently coupled at neutral pH or werecaptured by noncovalent methods. If a suitable epitopeor tag is not available, limited biotinylation of the kinaseat neutral pH for subsequent biotin capture offers analternative method of mild immobilization [19–21].

The second success factor was a carefully composedrunning and sample dilution buffer containing essentialcofactors (e.g., divalent metal ions) but simplified to ex-clude all nonessential components. This improved bufferenabled reliable analysis without interferences frombaseline drifts and unwanted background binding (Fig.2). The selected buffer composition was applicable toall kinases in the study, indicating similar features ofthe buffer requirements among kinases.

Workflow concept

We propose a step-by-step approach to kinase inhib-itor studies. The first step would involve selection ofbinders among a large number of compounds usingthe screening assay (Fig. 3). For previously uncharacter-ized binders, the activity/inhibition assay could then beused, in a second step, to establish whether the bindersare inhibitors of the enzyme. IC50 values could then bedetermined using concentration series of binders (Fig.4). The third step would then be a full kinetic character-ization of the selected compounds binding to the targetkinase (Fig. 5).

The importance of rate constants

Many lead optimization studies using a structure–ac-tivity relationship (SAR) or quantitative structure–activ-ity relationship (QSAR) approach use affinity-relateddata as the parameter for biological activity [22–24].Affinity is not the ideal parameter, however, because itis related to both of the binding rate constants(KD = kd/ka). Using ambiguous data such as KD andIC50 might confuse or conceal the relationship betweenactivity and structure. In contrast, the use of kinetic datadirectly links a change in structure to changes in recogni-tion (ka), stability (kd), or both—information that canimprove the lead optimization procedure [16]. Forp38a and CDK2, it was observed that the affinity varia-tion among the binders could be related to variations ofequal magnitude for both of the rate constants (Fig. 5).

This shows that the affinity differences between the bind-ers could be related either to each rate constant alone orto both rate constants concurrently. The kinetic rate con-stants can also be used to determine the thermodynamicreaction coordinates for inhibitor binding as reportedpreviously for p38a [11] and HIV-1 protease [25].

Qualitative binding assays

In addition to the main kinetic assays, binding experi-ments can give qualitative information, such as specific-ity, revealed by simultaneous detection of binding todifferent kinases or isoforms of kinases. This experimentalsetup used the configuration with two different immobi-lized molecules addressed simultaneously in the flow cellof Biacore S51 (Fig. 6). The binding ofATPand inhibitorsto activated and unactivated kinase could also be directlycompared using this setup (Fig. 8). This is an advantageover enzyme activity inhibition assays, where only bindersto the activated form of the kinase can be investigated.

If two or more binders of different kinetics are avail-able, these can be used in a chain of experiments map-ping overlapping binding sites on the target molecule.At high concentrations, ATP was able to transformthe apparent binding kinetics of inhibitors to Scheringkinase 3 and the activated p38a into ATP-like kinetics,revealing that the inhibitors were ATP competitors.

A broad range kinase study

The immobilization strategies and buffer conditionsthat were developed allowed the study of a broad rangeof kinases (Table 1). For most of the kinases, small mol-ecule inhibitor binding characterizations could be per-formed, yielding high-quality data. No generaldiscrepancies in the requirements and results betweentyrosine kinases and serine/threonine kinases wereobserved.

Conclusions

The methodology described in this article allowed usto study a broad range of kinases. The complementarymethods of screening and enzyme activity measurementsfacilitated the identification of binders and the charac-terization of their inhibiting properties. The direct bind-ing assay of small molecule inhibitors to the kinasesallowed the determination of the binding rate constants,resolving the affinities into their components. Qualita-tive assays demonstrated the specificity of the inhibitors,differences between ATP and inhibitor binding, bindingsite specificity, and differences in requirements for cofac-tors such as manganese ions.

This diverse and highly valuable information was ob-tained using label-free technology and suggests that the


methodology described here can be an extremely usefultool in the drug discovery process.

Acknowledgments

We thank Karsten Parczyk and Uwe Eberspacher ofSchering AG (Germany) and Barry Schweitzer of Pro-tometrix (USA) for providing valuable reagents to thestudy. We acknowledge Stefan Lofas, Bjorn Persson,Asa Frostell-Karlsson, and Anita Larsson of BiacoreAB (Sweden) for discussions.

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Kinetic studies of small molecule interactions with protein kinases using biosensor technology

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