A Biacore biosensor method for detailed kinetic binding analysis of small molecule inhibitors of p38alpha mitogen-activated protein kinase

Protein kinases are emerging as one of the most intensely studied classes of enzymes as their central roles in physiologically and clinically important cellular signaling events become more clearly understood. We report here the development of a real-time, label-free method to study protein kinase inhibitor binding kinetics using surface plasmon resonance-based biomolecular interaction analysis (Biacore). Utilizing p38alpha mitogen-activated protein kinase as a model system, we studied the binding properties of two known small molecule p38alpha inhibitors (SB-203580 and SKF-86002). Direct coupling of p38alpha to the biosensor surface in the presence of a reversible structure-stabilizing ligand (SB-203580) consistently produced greater than 90% active protein on the biosensor surface. The dissociation and kinetic constants derived using this Biacore method are in excellent agreement with values determined by other methods. Additionally, we extend the method to study the thermodynamics of small molecule binding to p38alpha and derive a detailed thermodynamic reaction pathway for SB-203580. The Biacore method reported here provides an efficient way to directly and reproducibly examine dissociation constants, kinetics, and thermodynamics for small molecules binding to p38alpha and possibly other protein kinases. Immobilization in the presence of a stabilizing ligand may further represent a broadly applicable paradigm for creation of highly active biosensor surfaces.     

Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities 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 fit 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 k(a), k(d), and K(D) 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 affinity 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 provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.     

Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods

The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors.     

Novel approach to analyzing RNA aptamer-protein interactions: toward further applications of aptamers

Surface plasmon-resonance analysis using a Biacore biosensor is a powerful tool for the detailed study of biomolecular interactions. The authors examined the methods of immobilizing proteins on the surface of NTA, SA, and CM5 sensor chips to study RNA aptamer-protein interactions. RNA aptamers and their deletion variants were loaded onto a protein-immobilized sensor chip, and their binding affinities were analyzed. Immobilizing the protein on a CM5 sensor chip via an anti-His-tag antibody was the only strategy that clearly detected the kinetic parameters of the interactions. DeltaNEO-III-14U, one of the deletion variants of the NS3 aptamer, had the highest binding affinity for the deltaNS3 protein in this study (KD = 4 x 10(-8)). Moreover, the 29-amino-acid spacer fragment was essential for protein immobilization using this strategy. This novel method will be useful in comparing the affinity of various RNA aptamers and selecting the most suitable candidates for a given target, as well as facilitating the in vitro selection procedure itself.     

Codeine-binding RNA aptamers and rapid determination of their binding constants using a direct coupling surface plasmon resonance assay

RNA aptamers that bind the opium alkaloid codeine were generated using an iterative in vitro selection process. The binding properties of these aptamers, including equilibrium and kinetic rate constants, were determined through a rapid, high-throughput approach using surface plasmon resonance (SPR) analysis to measure real-time binding. The approach involves direct coupling of the target small molecule onto a sensor chip without utilization of a carrier protein. Two highest binding aptamer sequences, FC5 and FC45 with K(d) values of 2.50 and 4.00 microM, respectively, were extensively studied. Corresponding mini-aptamers for FC5 and FC45 were subsequently identified through the described direct coupling Biacore assays. These assays were also employed to confirm the proposed secondary structures of the mini-aptamers. Both aptamers exhibit high specificity to codeine over morphine, which differs from codeine by a methyl group. Finally, the direct coupling method was demonstrated to eliminate potential non-specific interactions that may be associated with indirect coupling methods in which protein linkers are commonly employed. Therefore, in addition to presenting the first RNA aptamers to a subclass of benzylisoquinoline alkaloid molecules, this work highlights a method for characterizing small molecule aptamers that is more robust, precise, rapid and high-throughput than other commonly employed techniques.     

五种SPR传感芯片的再生制备及其应用

基于表面等离子体共振技术(surface plasmon resonance, SPR)的生物传感器,能够实时监测生物分子间的相互作用,且无需标记,已被广泛应用于蛋白质组学、药物研发、临床诊断、食品安全和环境监测等领域,并且显示出广阔的应用前景。传感芯片是Biacore系列仪器的核心部件,目前芯片只能从Biacore公司购买,价格昂贵,导致很多仪器利用率低下,资源处于闲置状态。阐述了用于Biacore系列仪器的五种传感芯片（J1,C1,CM5,SA和NTA芯片）的再生制备方法,并列举了应用实例,制备方法操作简单,成本低廉。通过多年的改进与优化,制备的芯片能够达到Biacore芯片同等品质。此方法的推广,将有助于推动表面等离子共振技术在各个领域的广泛应用。     

一种葡聚糖芯片的再生方法、表征及其应用*

表面等离子体共振（surface plasmon resonance, SPR）生物传感器,作为一种适时快捷,无需标记的生物分子相互作用研究工具,已广泛应用于生物化学分析与研究。羧甲基化葡聚糖修饰的CM5传感芯片是Biacore 系列仪器应用最为普遍的核心部件,目前CM5芯片主要从法玛西亚公司购买,价格昂贵,且一旦共价交联的受体分子失活,就不能重复利用。阐述了一种简便、低成本、用于SPR生物传感器的葡聚糖修饰金膜芯片的再生方法及其表征和应用。用此方法再生的芯片能被循环伏安法和原子力显微镜很好地表征,并成功地用于抗前列腺特异性抗原(prostate-specific antigen,PSA)固定和PSA检测, 同时测定了PSA与其抗体之间的动力学和亲和常数。     

Effective rate models for receptors distributed in a layer above a surface: application to cells and Biacore

In the Biacore biosensor, a widely used tool for studying the kinetics of ligand/receptor binding, receptors are commonly localized to the sensor surface through attachment to polymers that extend from the surface to form a layer. The importance of the polymeric layer in analyzing data is controversial. The question of the effect of a binding layer also arises in the case of ligands interacting with binding sites distributed in the extracellular matrix of cells. To identify and quantify the effects of a binding layer on the estimation of association and dissociation rate constants, we derived effective rate coefficients. The expressions show that rate constants determined under the standard assumption that binding takes place on a two-dimensional surface underestimate the true reaction rate constants by a factor that depends on the ratio of the height of the layer to the mean free path of the ligand within the layer. We show that, for typical biological ligands, receptors, cells, and Biacore conditions, the binding layer will affect the interpretation of data only if transport of the ligand in the layer is slowed substantially--by one or two orders of magnitude--relative to transport outside the layer. From existing experiments and theory, it is not clear which Biacore experiments, if any, have transport within the dextran layer reduced to such an extent. We propose a method, based on the effective rate coefficients we have derived, for the experimental determination of ligand diffusion coefficients in a polymeric matrix.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods

Two biophysical methods, Biacore and KinExA, were used to kinetically and thermodynamically characterize high-affinity antigen/antibody complexes. Three to five independent experiments were performed on each platform with three different antigen/antibody complexes possessing nanomolar to picomolar equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for 4 h, we were able to measure dissociation rate constants (kd) on the order of 1 x 10(-5)s(-1). To characterize high-affinity interactions by KinExA, samples needed to be equilibrated for up to 35 h to reach equilibrium. In the end, we show that similar kinetic rate constants and affinities were determined with both solution-phase and solid-phase methodologies. These results help further validate both interaction technologies and illustrate their suitability for characterizing extremely high-affinity interactions.     

Integration of surface plasmon resonance with mass spectrometry: automated ligand fishing and sample preparation for MALDI MS using a Biacore 3000 biosensor.

Integrating surface plasmon resonance analysis with mass spectrometry allows detection and characterization of molecular interactions to be complemented with identification of interaction partners. We have developed a procedure for Biacore 3000 that automatically performs all steps from ligand fishing and recovery to sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry including on-target digestion. In the model system used in this study a signal transduction protein, calmodulin, was selectively captured from brain extract by one of its interaction partners immobilized on a sensor chip. The bound material was eluted, deposited directly onto a MALDI target, and analyzed by mass spectrometry both as an intact protein and after on-target tryptic digestion. The procedure with direct deposition of recovered material on the MALDI target reduces sample losses and, in combination with automatic sample processing, increases the throughput of surface plasmon resonance mass spectrometry analysis.     

Interactions of acid sphingomyelinase and lipid bilayers in the presence of the tricyclic antidepressant desipramine

The tricyclic antidepressant desipramine causes a decrease in cellular acid sphingomyelinase (A-SMase, EC 3.1.4.12) activity when added to culture medium of human fibroblasts. This effect can be prevented by incubation of the cells with the protease inhibitor leupeptin, which suggests that desipramine induces proteolytic degradation of the lysosomal enzyme. By using surface plasmon resonance (SPR, Biacore) we were able to monitor the interactions of A-SMase and substrate-containing lipid bilayers immobilized on the surface of a Pioneer trade mark L1 sensor chip. SPR binding curves show that the enzyme hardly dissociates from the lipid surface at acidic pH values. On the other hand, a drop in binding signals (resonance units, RU) of approximately 50% occurred after injection of 20 mM desipramine. Our findings indicate that desipramine interferes with the binding of A-SMase to the lipid bilayers and thereby displaces the enzyme from its membrane-bound substrate. The application of control substances suggests a key role for the cationic moiety of desipramine. We hypothesize that the displacement of the glycoprotein A-SMase from the inner membranes of late endosomes and lysosomes by desipramine renders it susceptible to proteolytic cleavage by lysosomal proteases.     

Real-time analysis of antibody binding interactions with immobilized E. coli O157:H7 cells using the BIAcore

The kinetic properties of antibody-antigen reaction and other interacting macromolecules can be analyzed in real-time using the surface plasmon resonance biosensor (BIAcore). The interactions of an antibody against Escherischia coli O157:H7 were studied using immobilized whole cells. The bacterial sensor surface was evaluated with anti-E. coli in a continuous flow system. Regeneration of the sensor surface with guanidine-HCl was more effective than with phosphoric acid and resulted in better binding reproducibility. The determined kinetic values, association and dissociation rate constants, can be used in the development of rapid immuno-techniques. This study also provides the basis to evaluate real-time interactions of macromolecules with immobilized cells.     

Kinetic analysis of the binding of Ulex europeas agglutinin 1 (UEA 1) to H-antigenic fucolipid

The kinetics and thermodynamics of Ulex europeas agglutinin 1-H-antigenic determinant was studied by monitoring the binding of the lectin to H-antigenic fucolipid incorporated in liposomes tethered to the sensor surface by surface plasmon resonance. The second-order rate constants for the interactions are several order of magnitude slower than the diffusion controlled reactions. Analyses of activation parameters reveals that these slow second-order rate constants are a consequence of an unfavorable activation enthalpy term.     

Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet

ForteBio’s Octet optical biosensor harnesses biolayer interferometry to detect and quantify molecular interactions using disposable fiber-optic biosensors that address samples from an open shaking microplate without any microfluidics. We recruited a monoclonal antibody against a panel of peptides to compare the Octet directly with Biacore’s well-established 3000 platform and Bio-Rad’s recently launched ProteOn XPR36 array system, which use surface plasmon resonance (SPR) to detect the binding of one analyte over four surfaces and six analytes over six surfaces, respectively. A sink method was used to prevent analyte from rebinding the ligand-coated Octet tips and enabled us to extract accurate kinetic rate constants, as judged by their close agreement with those determined by SPR. Although the Octet is not sensitive enough to detect the binding of small molecules directly, it can access their affinities indirectly via solution competition experiments. We conducted similar experiments on the SPR instruments to validate these measurements. The Octet is emerging as a versatile complement to other more sophisticated biosensors, and the ProteOn provides high-quality data near the sensitivity of Biacore but in a more multiplexed format. Our results provide a benchmark for assessing the performance of the above-mentioned sensors.     

Kinetic screening of antibodies from crude hybridoma samples using Biacore

Experimental and data analysis protocols were developed to screen antibodies from hybridoma culture supernatants using Biacore surface plasmon resonance biosensor platforms. The screening methods involved capturing antibodies from crude supernatants using Fc-specific antibody surfaces and monitoring antigen binding at a single concentration. After normalizing the antigen responses for the amount of antibody present, a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants (k(a) and k(d)) and affinity (K(D)) could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of antigen were performed to validate the reliability of single-concentration measurements. The screening protocols can be used to characterize antigen binding kinetics to approximately 200 antibody supernatants per day using automated Biacore 2000 and 3000 instruments.     

A Theoretical and Experimental Study of Competition Between Solution and Surface Receptors for Ligand in a Biacore Flow Cell

Rate constants that characterize the kinetics of binding and dissociation between biomolecules carry fundamental information about the biological processes these molecules are involved in. An instrument that is widely used to determine these rate constants is the Biacore. In a Biacore experiment, one of the reactants, which we will call the receptor, is immobilized on a sensor chip. During the binding phase of the experiment the other reactant flows past the chip. After binding, buffer alone is introduced into the flow cell and dissociation is monitored. Often surface-based binding assays are influenced by the transport of the reactant in solution, complicating the determination of the chemical rate constants from the observed binding kinetics. We propose a new way to determine the dissociation rate constant by adding soluble receptor during dissociation. The method is tested first on simulated data and then on Biacore experiments where the lac repressor protein binds and dissociates from a stretch of double stranded DNA containing the lac repressor binding site. With this method we find a dissociation rate constant k_d=0.075 ± 0.005s^-1, a value that is faster than previously obtained from Biacore experiments. In developing our method to analyze these experiments we obtain an expression for the transport limited rate constant for a Biacore experiment when soluble receptor is present during dissociation.     

Comparison of biosensor platforms in the evaluation of high affinity antibody-antigen binding kinetics

The acquisition of reliable kinetic parameters for the characterization of biomolecular interactions is an important component of the drug discovery and development process. While several benchmark studies have explored the variability of kinetic rate constants obtained from multiple laboratories and biosensors, a direct comparison of these instruments' performance has not been undertaken, and systematic factors contributing to data variability from these systems have not been discussed. To address these questions, a panel of ten high-affinity monoclonal antibodies was simultaneously evaluated for their binding kinetics against the same antigen on four biosensor platforms: GE Healthcare's Biacore T100, Bio-Rad's ProteOn XPR36, ForteBio's Octet RED384, and Wasatch Microfluidics's IBIS MX96. We compared the strengths and weaknesses of these systems and found that despite certain inherent systematic limitations in instrumentation, the rank orders of both the association and dissociation rate constants were highly correlated between these instruments. Our results also revealed a trade-off between data reliability and sample throughput. Biacore T100, followed by ProteOn XPR36, exhibited excellent data quality and consistency, whereas Octet RED384 and IBIS MX96 demonstrated high flexibility and throughput with compromises in data accuracy and reproducibility. Our results support the need for a “fit-for-purpose” approach in instrument selection for biosensor studies.     

Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.     

Surface plasmon resonance for probing quadruplex folding and interactions with proteins and small molecules

Surface plasmon resonance is a technique for detecting binding events at the surface of a thin metal film. Through the commercial availability of instrumentation and sensor chips, the technique has found widespread application for determining the affinity and kinetics of macromolecular interactions. A variety of quadruplex forming oligonucleotides have been immobilized to sensor chips to permit analysis of their binding interactions with both small molecule and protein analytes. The fold of the quadruplex must be maintained through an appropriate choice of buffer, and care must be taken to ensure that data interpretation is not hampered by non-specific binding and adsorption of the analyte to the sensor surface and instrument. Affinity constants determined by surface plasmon resonance for interactions with quadruplexes correlate meaningfully with other methods, such as UV-visible and fluorescence titrations, enzyme linked immunosorbent assay, thermal melting studies and telomerase inhibition. Kinetic measurements of the association and dissociation of duplexes of quadruplex forming oligonucleotides and their complementary strands have enabled calculation of the folding and unfolding rates of the quadruplex itself, and determination of its stability as a function of buffer composition.