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Analytical Biochemistry 359 (2006) 94–105 www.elsevier.com/locate/yabio
Comparative analysis of 10 small molecules binding to carbonic anhydrase II by diVerent investigators using Biacore technology
Giuseppe A. Papalia a, Stephanie Leavitt b, Maggie A. Bynum c, Phinikoula S. Katsamba a, Rosemarie Wilton d, Huawei Qiu e, Mieke Steukers f, Siming Wang g, Lakshman Bindu h, Sanjay Phogat i, Anthony M. Giannetti j, Thomas E. Ryan k, Victoria A. Pudlak k, Katarzyna Matusiewicz l, Klaus M. Michelson m, Agnes Nowakowski n, Anh Pham-Baginski o, Jonathan Brooks p, Bryan C. Tieman q, Barry D. Bruce r, Michael Vaughn r, Michael Baksh s, Yun Hee Cho t, Mieke De Wit u, Alexandra Smets u, Johan Vandersmissen u, Lieve Michiels u, David G. Myszka a,¤
Center for Biomolecular Interaction Analysis, School of Medicine, University of Utah, Salt Lake City, UT 84132, USA b Gilead Sciences, Foster City, CA 94404, USA c Agilent Technologies, Palo Alto, CA 94304, USA d Argonne National Laboratory, Argonne, IL 60439, USA e Genzyme Corporation, Framingham, MA 01701, USA f Dyaxsa, B-4000 Liege 1, Belgium g Georgia State University, Marietta, GA 30302, USA h National Cancer Institute, Frederick, MD 21702, USA i National Institute of Allergies and Infectious Diseases, Bethesda, MD 20892, USA j Roche, Palo Alto, CA 94304, USA k Reichert Analytical Instruments, Depew, NY 14043, USA l Adamed, 05-152 Czosnow, Poland m Amgen, Thousand Oaks, CA 91320, USA n diaDexus, South San Francisco, CA 94080, USA o Wyeth, Andover, MA 01810, USA p Wyeth, Cambridge, MA 02140, USA q Abbott Laboratories, Abbott Park, IL 60064, USA r University of Tennessee, Knoxville, TN 37996, USA s University of California, Berkeley, CA 94720, USA t Human Genome Sciences, Rockville, MD 20850, USA u Tibotec, B-2800 Mechelen, Belgium Received 20 July 2006 Available online 7 September 2006
Abstract In this benchmark study, 26 investigators were asked to characterize the kinetics and aYnities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be Wt to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the ka, kd, and KD parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak aYnity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study
Corresponding author. Fax: +1 801 585 3015. E-mail address: firstname.lastname@example.org (D.G. Myszka).
0003-2697/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2006.08.021
phosphate-buVered saline. Biochem. the instrument was primed with water. Thus. an enzyme responsible for the conversion of carbon dioxide to bicarbonate. S51. We found an excellent correlation between the two methods across the wide range in aYnities exhibited by these compounds. buVer reagents. come some challenges.A. and 96-well plates. 30 SPR biosensor users illustrated that the aYnity determined from the biosensor for a small molecule/enzyme interaction matched the value determined in solution using isothermal titration calorimetry (ITC) . once using water. Then. Sensor chips. Instrument cleaning Before beginning the experiment. DMSO. and future drugs targeting this enzyme may lead to treatments for cancer and obesity . Our results highlight the strengths of biosensor analysis but also point to some challenges. Uppsala.5% (w/v) sodium dodecyl sulfate (SDS). and a maintenance chip was docked. In our Wrst comparative study. EDC. The current study builds on this previous work and is modeled on a growing biosensor application in a drug discovery setting: the rapid and reliable determination of kinetic and aYnity constants for a small panel of compounds binding to a single protein target. Papalia et al. Materials and methods Instrumentation and reagents Interaction analyses were performed using Biacore 1000. . SDS. With the wide array of opportunities oVered by this technology. This information is becoming crucial to better rationalizing compound behavior in bioassays and helping to steer compound selection and design strategies. sodium dodecyl sulfate. N-hydroxysuccinimide. The instrument was then primed Wve times using 0. with regard to what we can expect to see in terms of the variability of results within a given experimental system. Carbonic anhydrase isozyme II (CAII) from bovine erythrocytes. Surface plasmon resonance.5). SPR There is growing interest in applying optical biosensor technology. surface plasmon resonance. Kinetics. 2000. such as surface plasmon resonance (SPR)1based Biacore instruments. and T100 instruments (Biacore. 1 Abbreviations used: SPR. / Anal. Nethyl-NЈ-(3-dimethylaminopropyl)carbodiimide (EDC). N-ethyl-NЈ-(3dimethylaminopropyl)carbodiimide. as well as the level of experimental variation. The results of this test established experimental and data processing protocols for mass transport-limited reactions. based on our yearly review of the optical biosensor literature [1–6]. dimethyl sulfoxide. For the past few years. These results conWrmed that immobilization of an enzyme onto the sensor surface does not necessarily aVect the binding constants as is often presumed by critics of the biosensor approach. we organized a study with 36 participants who analyzed the interaction of a small molecule/macromolecular target system having a very fast association rate . MO. coeYcient of variation. however. All participants in this study were provided the same reagents and were asked to follow the same experimental protocol. carbonic anhydrase isozyme II. we have been addressing both of these issues by coordinating intra. We found that a majority of the participants were able to resolve the binding constants for all 10 compounds. For example. Wve times using 50 mM glycine (pH 9. We did. Current inhibitors of this enzyme are used to treat diseases such as glaucoma and epilepsy. Keywords: Biacore. PBS. RU. © 2006 Elsevier Inc. Sweden).Comparative analysis using Biacore technology / G. N-hydroxysuccinimide (NHS). Together. USA). We also compared the equilibrium dissociation constants obtained for the 10 compounds with those determined by ITC. each participant was asked to perform a series of instrument cleaning steps.and inter-technology benchmark studies. Louis. as well as sampling vials. as well as the general scientiWc community. that one would expect to observe when using Biacore technology for small molecule analyses. however. we coordinated a study with 22 users resolving the binding constants for a highaYnity monoclonal antibody–antigen interaction . 359 (2006) 94–105 95 provide insight into the challenges. ITC. identify a suboptimal subset of data (sensorgrams that were marred by features such as spikes and drift) and we explored the causes of these features. The goal of the current biosensor study was to determine the consistency of the results for small molecule analysis and to discover ways to further improve the application. And based on comments we receive from manuscript reviewers and participants at scientiWc meetings. isothermal titration calorimetry. the previously used chip was undocked. we Wnd that there is still a need to train investigators in how to design and perform biosensor experiments as well as in how to properly process and analyze data. caps. the 10 sulfonamide inhibitors. First. and general laboratory supplies were purchased from Sigma–Aldrich (St. All rights reserved. these benchmark studies educated the users of biosensor technology. dimethyl sulfoxide (DMSO). and ethanolamine HCl. in drug discovery. 3000. were obtained from Biacore. Protein–protein interaction. response units. CAII. and once again using water. CV. NHS. Recently. The target protein for this work was carbonic anhydrase II. This work trained users in how to generate and analyze data for a slowly dissociating system. we see that some skepticism remains regarding the validity of the binding constants determined using biosensors. the choice of carbonic anhydrase as the target represents a realistic model for small molecule drug studies.
4 (§)-Sulpiride 4-(Aminomethyl)benzenesulfonamide hydrochloride hydrate Sulfanilamide Furosemide 4-Carboxybenzenesulfonamide Dansylamide 1.4.1 M NHS and 0. Samples and buVer blanks were aliquotted in 7-mm Biacore vials. Then 70 ml of this 10£ PBS was added to 630 ml of degassed deionized water to make 1£ PBS buVer.3-Benzenedisulfonamide Benzenesulfonamide 7-Fluoro-2. a DMSO calibration series was prepared by adding 15 l of DMSO and 150 l of water to separate 1.9) after the biosensor was primed with immobilization buVer and equilibrated to 25 °C. Then 18 ml of DMSO (also provided to the participant) was added to 600 ml of the 1£ PBS buVer. 100 l of each compound at the highest concentration was added to 400 l of running buVer with 3% DMSO and this dilution was repeated. This was followed by injection of 0. and this buVer was Wltered. Three lower concentrations of each of the 10 compounds were then prepared via Wvefold serial dilutions into running buVer with 3% DMSO.4 0. pH 7. water (0.016. we chose to leave Xow cell 2 unmodiWed as a reference surface. 2000. 30 l of each compound solution was added to 1 ml of sample preparation buVer with no DMSO and mixed thoroughly. and 0.96 Comparative analysis using Biacore technology / G. and 0 l of the “+” solution were dispensed into Eppendorf tubes labeled d1 to d5. 100. The remaining 100 ml of 1£ PBS was divided into two 50-ml portions. dispensed into the 96-well plate. Users of S51 platforms activated the surfaces via sequential injections of NHS and then EDC for 7 min. 0. and the other was set aside as “sample preparation buVer with no DMSO” for use in analyte preparation.l injections each of 100 mM HCl. each of the compounds analyzed in this study was assigned a number (Table 1). BuVer preparation Participants were provided a 10£ phosphate-buVered saline (PBS. 2 0. 10 0. 2. 1£ D 20 mM Na2HPO4–NaH2PO4. 0.08. 6900 response units (RU) of CAII were immobilized. 100.4) stock solution. the chip was preconditioned with two 10. Biacore S51and T100-based analyses used 96-well plates for sampling. Dilutions were performed directly in the plates by taking 50 l from wells containing 250 l of the highest analyte concentration and diluting successively into wells containing 200 l of running buVer with 3% DMSO. Table 1 Compounds examined in this study Sample 1 2 3 4 5 6 7 8 9 10 Compound Analyte preparation For simplicity. 10 0. To prepare the samples for analysis.08.25 mg/ml CAII was injected for 7 min. and 400 l of the “¡” solution were added to tubes d1 to d5 so that each tube contained a Wnal volume of 400 l. which were then capped with soft rubber caps (BR-1005-55) that reseal after puncture to minimize evaporation. By making the start of each injec- Molecular mass (Da) 341 223 172 331 201 250 236 157 217 222 Concentration ( M) 2. 0. Instrument optimization To create a slight mismatch in the refractive index of the running buVer and sample solutions. 359 (2006) 94–105 Chip preconditioning After docking the CM5 sensor chip and priming the instrument with running buVer. 300.4.08. Then 400. 300. Enzyme immobilization Participants were provided with aliquots of 100 g of lyophilized CA II that were centrifuged and dissolved in 400 l of 10 mM sodium acetate (pH 4. Surfaces were then blocked via a 7-min injection of ethanolamine at a Xow rate of 10 l/min. DMSO is commonly used as a solvent for small molecule studies.97%.4 M EDC.08.3-benzoxadiazole-4-sulfonamide Acetazolamide . Although the Wnal concentration of DMSO in this buVer via this addition is actually 2. 2 0. 10 0. for Biacore S51 and T100 analyses. 150 mM NaCl. 200. 10 0.25 mg/ml CAII for 7 min at a Xow rate of 10 l/ min. and 3000. 2. Preparation of analyte in this manner ensures that the concentration of DMSO is matched with that of running buVer with 3% DMSO. 200. Participants were provided with a stock solution of each compound dissolved in DMSO. 2.4. 50 mM NaOH. and residual activated groups on the surface were blocked by a 7-min injection of 1 M ethanolamine (pH 8. 50 0. Using these sample volumes and the resealable caps permitted the three concentrations of each compound to be sampled twice. and 0.A.4% of the total volume of running buVer) was added to the running buVer with 3% DMSO.4.1. Using a Xow rate of 20 l/min. For this set of studies. One of these portions was set aside for use as “immobilization buVer”.5-ml aliquots of running buVer with DMSO to obtain “+” and “¡” solutions. / Anal. Papalia et al. 0. To correct for the excluded volume eVect.016. The Wve DMSO calibration solutions were either aliquotted into 7-mm Biacore vials or. 10. for simplicity this buVer is termed “running buVer with 3% DMSO”. 50 2. 0. 0. Biochem.5% SDS at a Xow rate of 100 l/min. 10. For analyses using Biacore 1000.5). On average.4. 2. 0.4. the surface of Xow cell 1 was activated for 7 min using a mixture of 0.
Australia). Analytes were sampled twice using Biacore 1000. and T100.l injection of running buVer was also included as an additional wash step). The analyte injection was followed by an ExtraClean wash command that automatically Xushes the sample delivery system with running buVer (for Biacore 2000 and 3000. S51. and 3000 and were sampled once using Biacore S51 and T100.4. 359 (2006) 94–105 97 tion easily identiWable. Means and standard deviations were computed using Microsoft Excel software. Campbell. a 15. . 2. The instrument was primed three times with this spiked running buVer and then normalized using 40% (v/v) glycerol for Biacore 1000 and 2000 or 70% (v/v) glycerol for Biacore 3000. Double-referenced  association and dissociation phase data for compounds 1 to 8 were globally Wt to a simple 1:1 interaction model (A + B D AB). 0. Finally. The experimental error or coeYcient of variation (CV) was calculated as (Standard Deviation/Mean) £ 100. The DMSO calibration series was then injected. Data shown are from the analysis of compound 4 by participant H and were collected using a Biacore 2000 instrument. 2.Comparative analysis using Biacore technology / G. / Anal. High-quality kinetic data set. Compound was diluted in the same buVer as protein to minimize heat of C A B D E F Fig. the individual compound 50 samples were tested (from lowest to highest concentrations within each series) and each compound series was separated by a blank buVer injection. During each binding cycle. Compounds 9 and 10 were Wt to a 1:1 interaction model that included a mass transport term (Ao D A + B D AB) . Papalia et al. (D) Double-referenced response not described by a simple exponential. (E) Dip below baseline for double-referenced response. Biochem. analyte was injected for 1 min at a Xow rate of 100 l/min (90 l/min for Biacore S51) and dissociation was monitored for 140 s. Problematic sensorgrams. (A) SigniWcant spikes at the start of the association and dissociation phases. Duplicate analyses of 0. this mismatch aids in data processing . 2000. (F) Refractive index jumps.0. Analyte injection order and analysis method Data were collected at the highest collection rate possible and at 25 °C. 1. Isothermal titration calorimetry Protein was dissolved in 1 £ PBS and buVer exchanged to minimize any contaminants from lyophilization. and 10 M furosemide binding to 6450 RU of immobilized CAII. (C) BuVer blank responses that do not overlay. followed by two additional blank injections. An initial series of buVer blanks was injected Wrst to fully equilibrate the system. (B) Spikes within a sensorgram.A. Data processing and kinetic analysis Data sets were processed and analyzed using Scrubber 2 (BioLogic Software. Response (RU) 25 0 0 60 120 180 Time (s) Fig.
2. Isothermal titration calorimetry experiments were performed using a Microcal VP-ITC instrument (MicroCal. Data were analyzed with a single-site binding model using the Origin software provided by MicroCal. Northampton. USA) . and 400 M for compounds 1 to 4. Experiments were performed with the compound in the syringe and the protein in the cell. (For interpretation of the references to color in this Wgure legend.l injections of compound into protein with 240 s between each injection and a stirring speed of approximately 300. 600. and 300 M for compounds 9 and 10. MA. participants used Biacore biosensor technology to characterize the interactions of 10 compounds with the enzyme CAII. The heat of dilution was taken either from the points after saturation. Data sets obtained using Biacore 1000 (A) and Biacore 2000 (B–I) instruments. 65. the stoichiometry value was set to 1 assuming a 1:1 ratio. which has been well established for this system.) . Results In this benchmark study. the weakest inhibitor. 3. Protein concentration was measured using an extinction coeYcient at A280 of 50. Biochem. control experiment of compound into buVer. To minimize the dependency of the Wtting parameters for compound 1. / Anal. Experiments were performed at 25 ° C. We provided each participant 6 7 8 9 10 2 3 4 5 B C D Response (RU) E F G H I 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 Time (s) Fig.98 Comparative analysis using Biacore technology / G. The concentrations of compounds titrated were 960. Columns 1 through 10 correspond to the numbering of compounds in Table 1. and 38 M for titrations of compounds 1–3 and 28 M for titrations of compounds 4–10. with an initial Wrst injection of 3 l and subsequent 10. 359 (2006) 94–105 dilution eVects from buVer mismatch. 350 M for compounds 5–8.070 L mol¡1 cm¡1. Experimental parameters included: 30 injections of compound. Responses shown in red exhibited one or more of the characteristics described in Fig. Corresponding concentrations of carbonic anhydrase were 44. respec1 A tively.A. Papalia et al. or from a minimization of the 2 value. 666. the reader is referred to the Web version of this article.
whereas the Biacore S51 and T100 users performed a single injection of each. the biosensor was performing optimally. and buVer. / Anal.A. we can tell that the experiment was designed and executed properly. Each concentration series was bracketed by buVer blanks that were included for double referencing . From inspection of this data set. Immobilization densities ranged from 2400 to 13. Three concentrations of each compound. 359 (2006) 94–105 99 with a detailed experimental protocol as well as stock solutions of the enzyme.Comparative analysis using Biacore technology / G. Hallmarks that 6 7 8 9 10 2 3 4 5 K L M Response (RU) N O P Q R S 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 Time (s) Fig. Columns 1 through 10 correspond to the compound numbering given in Table 1. were analyzed for CAII binding at 25 °C. 1. An example of a good data set obtained for one compound is shown in Fig. Good and bad sensorgrams To illustrate how we interpreted the responses generated by the diVerent users. prepared in a Wvefold dilution series. Data sets obtained using Biacore 3000 (J–S) instruments. Biochem. 4. Papalia et al. and 3000 platforms did repeat injections of each analyte concentration. and the obtained responses are reliable. . Because each compound dissociated completely from the CAII surface within minutes after the end of the association phase. we Wrst highlight examples of good and bad data. 2000.000 RU. 1 J Participants using Biacore 1000. Responses shown in red exhibited one or more of the characteristics described in Fig. no regeneration step was required. CAII was immobilized on one Xow cell surface of a CM5 sensor chip using standard amine-coupling chemistry and an unmodiWed Xow cell surface served as a reference. 2. this variation was useful in determining whether there were any systematic trends in the results with surface density. compounds.
and in some cases drop below the baseline during the dissociation phase (Fig. Data sets obtained using Biacore S51 (T–Z) and Biacore T100 (AA) instruments. / Anal. And when data sets are of poorer quality (e. inconsistencies in buVer injections may indicate sample carryover. 359 (2006) 94–105 make this data set a good one include responses that are concentration dependent. Although it 1 T is common to have a few data points producing spikes at the transitions between running buVer and the analyte sample. 2. 2C would make it diYcult to choose which blanks to use for double referencing. Sensorgrams that. after referencing. The inability to achieve steady state after a fast association phase (Fig. 2B) often are indicative of a poorly degassed buVer or a dirty injection system. Frequent spikes throughout a sensorgram or sets of sensorgrams (Fig. Responses shown in red exhibited one or more of the characteristics described in Fig. the reader is referred to the Web version of this article. Also. the anomalies and/or complexity most often can be eliminated by optimizing the assay design.g. 2E). When data sets are of high quality like the one shown in Fig.A. Papalia et al. when they exhibit unusual proWles or can be described only by a complex model). the biosensor’s Xuidic system. the quality of some of the data sets generated by our study participants was not on par with the example shown in Fig.g.. For example. well-behaved 1:1 interactions for this study. they often can be described by a 1:1 interaction model. may result from nonspeciWc binding to the reference surface. participant Z was unable to collect data for compound 8. but the not-reproducible responses from the replicate buVer blanks shown in Fig.100 Comparative analysis using Biacore technology / G. replicate injections that overlay. Biochem. 2. 1.. Six of the common anomalous sensorgram features we observed in some data sets are depicted in Fig. Blanks should have similar proWles and generally overlay. 2A) indicate poor-quality injections that may be caused by a fouled integrated Xuidic cartridge. 2D) in a system known to be 1:1 suggests that there may be problems with reagent preparation. signiWcant spikes at the both the beginning and end of the association phase apparent after referencing (Fig. 5. or both. (For interpretation of the references to color in this Wgure legend. >2 s) can result in the loss of kinetic information when the binding responses are fast. 1. Columns 1 through 10 correspond to the compound numbering given in Table 1. Sustained jumps in refractive index 6 7 8 9 10 2 3 4 5 U V W Response (RU) X Y Z AA 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 Time (s) Fig. Due to a lack of compound.) . dip down during the association phase. spikes that occur over a larger window of time (e. Although we intentionally chose well-characterized. and clearly discernible exponential curvature during both the association and dissociation phases.
2 were highlighted in the participants’ data sets. Sensorgrams that displayed any of the characteristics illustrated in Fig. Visual inspection of participants’ data All of the data sets submitted by the 26 participants are shown in Figs. SigniWcantly Xawed sensorgrams were omitted from the kinetic analysis. and S were excluded entirely.Comparative analysis using Biacore technology / G. the 1 B consistency in the binding proWles demonstrates the similarity between data sets obtained for a single compound from diVerent participants. 6 7 8 9 10 2 3 4 5 C D E F Response (RU) G H I L M Q 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 Time (s) Fig. 6. / Anal.A. Biochem.) . 2F) make obtaining reasonable Wts impossible without elaborate data processing. Individual data sets omitted from the kinetic analysis are indicated by red Xs. Papalia et al. K. R. Binding responses (black lines) are overlaid with the Wt of a 1:1 interaction model (red lines). whereas data panels from participants A. P. whereas those shown in red exhibited one or more problems. the diVerent binding proWles demonstrate that the 10 compounds bind the target with diVerent aYnities. Across each row. J. and responses for compounds 9 and 10 were Wt to a 1:1 model that included a mass transport term. the reader is referred to the Web version of this article. N. Kinetic analysis of data sets shown in Figs. Down each column in these Wgures. 3–5. (For interpretation of the references to color in this Wgure legend. Rate constants determined from this analysis are summarized in Table 2. 3–5. Responses for compounds 1 to 8 were Wt to a simple 1:1 model. 359 (2006) 94–105 101 (Fig. Sensorgrams considered to be acceptable for kinetic analysis are shown in black. O.
the responses are of excellent quality. we do not know the cause of the high number of failed sensorgrams obtained from these and the other Biacore 3000 instruments. These results suggest that the problem might lie elsewhere in these systems. 4). changes in bulk refractive during the course of the injection could not be referenced properly. respectively. Biochem. and 96% of these sensorgrams were included in the kinetic analysis. At this time. Data sets B–I were obtained using Biacore 2000 instruments (Fig. Much of the data collected from the Biacore 3000 instruments were compromised in quality. Interestingly. 9. 3). but other deviations in the sensorgrams require the ability to double reference the data. data sets Q and S (Fig. 4) were obtained using Biacore 3000 instruments after servicing that included replacing the integrated Xuidic cartridge. and only 18% of these sensorgrams were included in the kinetic analysis. Overall. 3). Data sets J–S were obtained using Biacore 3000 instruments (Fig. It is surprising that it is possible to detect binding responses for small molecules using the Biacore 1000. and 90% of these sensorgrams were included in the kinetic analysis. Due to the absence of an in-line reference Xow cell in Biacore 1000. / Anal. Common features include large spikes at the beginning and end of the association phase as well as spikes throughout the data sets. and yet the data are still of poor quality. Given the challenges in working with this older system. 6 (continued) Data set A was obtained using a Biacore 1000 instrument (Fig. In some cases. Kinetic analysis Data sets collected for compounds 1–8 were Wt to a simple 1:1 model.A. Data sets T–Z and AA (Fig. and 10. it is interesting to note that even these very early versions of Biacore technology were capable of small molecule detection at the qualitative level. it is possible to model the bulk shift. and data sets for compounds 9 and 10 were Wt to a 1:1 model that included a term for mass transport . 5) were obtained using Biacore S51 and Biacore T100 instruments.102 Comparative analysis using Biacore technology / G. 359 (2006) 94–105 1 T 2 3 4 5 6 7 8 9 10 U V W Response (RU) X Y Z AA 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 0 45 90 Time (s) Fig. participant A focused on the analysis of the highest aYnity compounds: 8. Although the data might not be as clean as those with more recent instrument platforms. Papalia et al.
Fig.0 § 0.006 0.3 £ 104 6.11 § 0. 3–5 are displayed in Fig. we were Fig. Discussion A total of 26 participants contributed to this collaborative study of 10 compounds binding to CAII.083 § 0.01 0. Importantly. data points are tightly clustered and the CV in each of the binding constants is less than 40%: CV(ka) D 34%.80 § 0. ka versus kd plot for compounds 1–10 binding to CAII. In our review of the participants’ data sets.A. 8A shows the heat evolved as compounds 2.079 § 0.012 0. and CV(KD) D 37%.12 0.3 § 1. indicating a 1:1 stoichiometry for each interaction. IsoaYnity diagonals are shown as dashed lines.2 £ 104 2. the participants examined relatively few (three).1 § 1.1 1. all show a molar ratio of 1 at the midpoint of titration.013 0.2 0.031 KD ( M) 48 § 14 3.011 pounds have very fast association rates and are inXuenced by mass transport.17 0. 6.44 § 0. An above-average CV (32%) in kd for compound 1 is not unexpected given that the dissociation rate for this compound is very fast and therefore this set of sensorgrams contains less information than those in other data sets. However..037 § 0. The changes in one parameter are compensated for by changes in the other parameter.g.6 3. The KD values from the ITC analyses were plotted against those obtained using SPR (Fig.5 £ 104 3. Participants performed the analysis using Wve diVerent Biacore platforms. 4. KD D kd/ka) is not aVected during Wtting.7 § 0.. The aYnities determined from the two methods are highly correlated (99. 9). their ratio in fact leads to similar KD values. Biochem.74 0.97 § 0.7 § 1. In general. but widely diluted (Wvefold).03 0. Papalia et al.12 § 0.13 § 0.018 0.0 § 2. A larger experimental error in ka and kd for compounds 9 and 10 relate to the fact that these comTable 2 Rate and equilibrium constants determined from Biacore at 25 °C Sample 1 2 3 4 5 6 7 8 9 10 n 15 18 18 18 17 19 19 18 17 18 ka (M¡1 s¡1) 8. 7 for each compound corresponds to the variability in ka. The 10 compound–target interactions were also studied in solution using ITC. .6 § 0. 7).1 £ 106 kd (s¡1) 0.9 § 1.12 0.11 § 0. vertical. the cause of these discrepancies is not known. The spread in the constants determined for compounds 2 and 7 (e. respectively. 8B).061 § 0. The range of kinetic parameters derived for the 10 compounds is listed in Table 2 and illustrated in the kinetic distribution plot (Fig.4 £ 105 2. and KD reported by the panel of participants. CV(kd) D 24%. each compound–target interaction was well described by a 1:1 interaction model. 6. because under transport limited conditions the ka and kd values become coupled. and diagonal (perpendicular to the dashed lines) directions shown in Fig.4 £ 106 3. The Wts of the data sets shown in Figs.0 § 2.4 £ 104 3.8%).9 £ 105 1.3 £ 103 2.4 § 1.0 § 0. The titrations shown are representative of those observed for all of the compounds. The result is that the CVs for the KD of compound 9 (33%) and compound 10 (35%) compare favorably with the average for the 10 the compounds (CVavg(KD) D 37%). The spread of the data points in the horizontal. 7. so the ratio of the two (i. 359 (2006) 94–105 103 . with the greatest diVerence in KD between the two methods being only twofold for compound 6.Comparative analysis using Biacore technology / G.6 § 0. To balance sampling throughput and kinetic resolution.031 § 0.28 0.037 § 0.040 § 0.85 § 0. In general. CV(KD) D 47 and 87%) resulted from one and four outlier data sets.011 0.03 0.6 £ 105 1. kd. / Anal.38 § 0.1 § 0. The raw heats from these titrations were integrated to generate plots of kilocalories/mole of injected compound versus the molar ratio of compound and CAII (Fig. concentrations of each compound.02 0. and 8 were titrated into solutions of CAII.e.
This kinetic proWling provides critical information in a drug development setting. 359 (2006) 94–105 surprised by the large number of low-quality sensorgrams obtained using Biacore 3000 instruments (Fig. Initially.0 10-4 10-4 10-5 10-6 10-7 10-8 Molar Ratio Fig. KDITC (M) Fig. ITC analysis of compounds 1–10 binding to CAII. No improvement in data quality was observed between data sets R and S.5 2. 7) of the compounds provides insight into the variation one could expect to see for this type of analysis. (A) Representative titrations of compounds 2. the experimental error in the reaction parameters for all 10 compounds is small (average »30%). and this was expected because there is less information in these data sets. This observation. 5. we are pleased to see that the variation in the binding constants for all 10 compounds averaged approximately 30%. Papalia et al. . however. 9). the aYnity of CAII for these small molecules is not signiWcantly altered on immobilization in this manner.A. but these compounds are distinguishable based on their kinetic proWles. Rather than the surface itself producing erroneous binding constants. for example.0 2. These results suggest that the issue with running this assay using Biacore 3000 instruments may lie elsewhere. we thought that this was perhaps attributable to contaminated Xuidic cartridges. our experience suggests that sample heterogeneity and inappropriate experimental design and/or data analysis are the most common sources of reported discrepancies between solution. we do not know the source of the poorer quality data originating from Biacore 3000 instruments and whether or not it is speciWc to this particular set of samples. 4. 6. / Anal. The dissociation constants derived from the SPR and ITC studies of the 10 compounds were in excellent agreement (Fig. and we certainly KDSPR = KD ITC B -1 µcal/sec 10-8 kcal/mol of injectant cpd 1 data 10 -7 9 10 -5 2 4 5 KDSPR(M) 3 -9 6 7 8 10 -6 6 85 7 4 23 -13 9 10 10 -5 1 0. This was not unusual and helps to quantitate the types of variation one would expect to see in the assay. to some degree. This study also emphasizes the value of kinetic analyses compared with equilibrium-based analyses that yield only aYnity information. Clearly. We observed greater experimental error in the binding constants for weak interactions. At this time. Biochem. like the great majority of Biacore-based experiments.104 Comparative analysis using Biacore technology / G. given that two data sets were collected from Biacore 3000 instruments before (P and R) and after (Q and S) professional servicing during which the Xuidic cartridges were replaced. 8. along with the high degree of correlation observed for a number of other experimental systems [6.5 3. We also observed greater experimental error for A 0 25 Time (min) 50 75 100 0 25 Time (min) 50 75 100 2 6 4 8 compounds that were inXuenced by mass transport. particularly when one considers the challenges associated with analyzing small molecule interactions using SPR. This explanation seems unlikely. (B) Plots of kilocalories/mole of injectant versus molar ratio for each of the 10 compounds. The kinetic distribution plot (Fig. and 8. This is because these experiments.0 1. 4).5 1. deXates the criticism that the surface-based SPR method alters binding constants. involved the immobilization of ligand to a Xuid-like dextran layer attached to the gold surface and not to the gold surface directly.and surfacebased methods. in which an investigator wants to understand in detail how changes in a compound’s structure aVect its binding activity. We recognize that.14]. 9. and 8 are statistically identical. The dashed line depicts a correlation of 1. In general. this was an idealized study because each participant was provided with reagents and the experimental protocol. Correlation plot of aYnities determined for each of the 10 compounds using SPR and ITC. and only a partial improvement in sensorgram quality was observed between data sets P and Q. Overall. the CAII aYnities of compounds 4.
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