Measurement Of Binding Constants Research Articles

Theoretical analysis of the competition ligand‐based NMR experiments and selected applications to fragment screening and binding constant measurements

AbstractCompetition ligand‐based NMR experiments have recently emerged as a powerful approach for fragment‐based screening in the drug discovery process. The robustness and reliability of these methodologies result in the identification of only specific binding ligands. In addition, the experiments, when properly performed, provide a quantitative measure of the affinity of the identified hits for the receptor. This is relevant for ranking the molecules according to their binding strength to the biomolecular target and for building a meaningful structure activity relationship (SAR) table. Several NMR experiments have been proposed for this purpose. The theory at the base of these different competition ligand‐based NMR experiments is analyzed in detail and several simulations with experimental conditions typically used in the NMR screening experiments are reported. Most of the presented theory applies also to the direct ligand‐based NMR screening experiments. In addition, the strategy for deriving the binding constant of the molecules is described, and selected applications to relevant drug discovery projects are presented. Finally, a novel sensitive NMR screening experiment that combines WaterLOGSY and transverse relaxation effects is proposed. © 2008 Wiley Periodicals, Inc. Concepts Magn Reson Part A 32A: 341–372, 2008.

Investigation of Monovalent and Bivalent Enantioselective Molecular Recognition by Electrospray Ionization-Mass Spectrometry and Tandem Mass Spectrometry

In this work is described the investigation of bivalent versus monovalent enantioselective molecular recognition in the context of enantioselective separations. Electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) are used for evaluating enantioselective systems through the measurement of (1) relative solution-phase binding constants via titration and (2) relative gas-phase binding via collision threshold dissociation. In HPLC, a cinchonane-type chiral stationary phase (CSP) based on tert.-butylcarbamoylquinine provides vastly increased retention and enantioselectivity for separation of bivalent versus monovalent alkoxy-benzoyl-N-blocked leucine enantiomers. The bivalent enantiomers are able to span and simultaneously interact with multiple interaction sites on the CSP surface, leading to enhanced separation. ESI-MS titration measurements also show an increased avidity for binding between bivalent selector and bivalent selectand, compared with the monovalent system. However, enhanced enantioselectivities measured in HPLC for the bivalent system cannot be reproduced by MS due to inherent mechanistic differences. Assumed discrepancies in relative response factors also give rise to systematic errors which are discussed. The results of MS/MS gas-phase experiments show that enantioselectivity is essentially lost in the absence of solvation, but that dissociation thresholds can provide a measure of relative dissociation energy in the bivalent interaction system compared to the monovalent counterpart. Such measurements may prove useful and efficient in better understanding multivalent interactions, in line with current theoretical considerations of effective concentrations and ion trap effects. This is the first application of mass spectrometric methods for assessing increased avidity of binding in multivalent enantioselective molecular recognition.

4-N-, 4-S-, and 4-O-Chloroquine Analogues: Influence of Side Chain Length and Quinolyl Nitrogen pKa on Activity vs Chloroquine Resistant Malaria

Using predictions from heme-quinoline antimalarial complex structures, previous modifications of chloroquine (CQ), and hypotheses for chloroquine resistance (CQR), we synthesize and assay CQ analogues that test structure-function principles. We vary side chain length for both monoethyl and diethyl 4-N CQ derivatives. We alter the pKa of the quinolyl N by introducing alkylthio or alkoxy substituents into the 4 position and vary side chain length for these analogues. We introduce an additional titratable amino group to the side chain of 4-O analogues with promising CQR strain selectivity and increase activity while retaining selectivity. We solve atomic resolution structures for complexes formed between representative 4-N, 4-S, and 4-O derivatives vs mu-oxo dimeric heme, measure binding constants for monomeric vs dimeric heme, and quantify hemozoin (Hz) formation inhibition in vitro. The data provide additional insight for the design of CQ analogues with improved activity vs CQR malaria.

New Approach To Measure Protein Binding Based on a Parallel Artificial Membrane Assay and Human Serum Albumin

We report here a new, label-free approach to measure serum protein binding constants. The assay is able to measure HSA K d values in the milli-molar to micromolar range. The protein is not immobilized on any surface and the assay self-corrects for nonspecific adsorption. No mass balance is required to get accurate binding constants and it is not necessary to wait for equilibrium to extract the binding constant. The assay runs in a 96-well format using commercially available parts and is, therefore, relatively easy to implement and automate. As the chemical membranes used are not water permeable, there is no volume change due to the osmotic pressure and pretreatment (soaking) is not necessary. The concept can potentially be extended to other proteins and could thus serve as a label-free technique for general binding constant measurements.

Method of Measuring Oligonucleotide−Metal Affinities: Interactions of the Thrombin Binding Aptamer with K+ and Sr2+

We report a new, mass spectrometry-based method for measuring affinity constants for specific metal ion binding to DNA, particularly for quadruplex DNA. This method, which is applicable to other systems, utilizes the gas-phase signal fractions, as determined by mass spectrometry, from the bound and unbound species as input into a mathematical model that determines various parameters, one of which is the binding affinity constant. The system used to develop and test the model was the thrombin-binding aptamer, an appropriate quadruplex structure that binds both K+ and Sr2+ cations. Using this method, we measured the binding constants of potassium and strontium cations with the quadruplex structure to be 5000 and 240 nM, respectively. We then applied the method to measure the change in enthalpy of the binding of strontium cations to the thrombin binding aptamer. The DeltaH for this interaction is -71 kJ/mol (-17 kcal/mol). The binding constant measurements are consistent with earlier measurements on the same system, and the measured change in enthalpy is in excellent agreement with previous work.

Multivalence and spot heterogeneity in microarray-based measurement of binding constants

Microarray technology is increasingly used for a miniaturised and parallel measurement of binding constants. In microarray experiments heterogeneous functionalization of surfaces with capture molecules is a problem commonly encountered. For multivalent ligands, especially, however, binding is strongly affected by receptor density. Here we show that high-resolution imaging of microarrays followed by image segmentation and separate analysis of bright and dark parts provides valuable information about ligand binding. Binding titrations were conducted with monovalent and bivalent fluorescent ligand peptides for the model receptor vancomycin. Microarrays were scanned with a confocal microscope and inhomogeneous spots were evaluated either as a whole or after segmentation into bright and dark areas. Whereas the binding constant for the monovalent ligand was hardly affected by spot heterogeneity, for the bivalent ligand affinity was higher for the parts of the spots with a greater density of receptors. This information was lost if the spots were analysed as a whole. These results reveal that imaging resolution may be a key factor in miniaturised binding assays, emphasising the importance of high-resolution images and image segmentation for new techniques, for example SPR imaging.

Development of a multiplex non‐radioactive receptor assay: the benzodiazepine receptor, the serotonin transporter and the β‐adrenergic receptor

Binding assays still form a fundamental part of modern drug development. Receptor binding assays are mostly based on radioactivity because of their speed, ease of use and reproducibility. Disadvantages, such as health hazards and production of radioactive waste, have prompted the development of non-radioactive receptor binding assays. This application therefore focuses on measuring receptor-ligand interactions using mass spectrometry. Moreover, the novelty of this approach originates in determining multiple analytes in a single assay (multiplexing). The proof of principle of a non-radioactive multiplex receptor assay is demonstrated using a pool of receptors from rat cortical tissue with flunitrazepam, MADAM and pindolol in one vial with or without their respective displacers. Flunitrazepam, MADAM and pindolol bound specifically at 73%, 30% and 40% to their respective receptors. This corresponds to specific binding sites of 0.61 pmol/mg protein, 0.07 pmol/mg protein and 0.06 pmol/mg protein, respectively. We propose to measure the bound fraction instead of the free fraction in order to reach a significant difference in measured signals (total binding versus non-specific binding). The bound fraction can be obtained after dissociating the ligand from the receptor-ligand complex using 50% methanol in water. The current setup of the assay calls for further improvement with respect to the measurement of binding constants for a multitude of receptors in one assay with sufficient accuracy and precision.

Development of an ESI-MS screening method for evaluating binding affinity between integrin fragments and RGD-based peptides

Electrospray ionization mass spectrometry (ESI-MS) is used to evaluate binding affinities between integrin fragments and RGD-based peptide ligands. The integrin fragment peptides synthesized and studied were selected based on crystallographic and literature data which highlighted the specific binding pockets for the RGD motif in intact integrin proteins. Relative binding constants were determined by fitting the data obtained from ESI-MS titration experiments to a quadratic equation based on a 1:1 association model. Frontal analysis capillary electrophoresis (FACE) was used as a complementary solution phase technique to measure the absolute binding constants for the same host–guest systems and the results from both techniques were compared. Gas phase collision activated dissociation threshold measurements in an ion trap mass spectrometer also were carried out on the complexes to further validate binding constant measurements. Better correlation was observed between the MS/MS and CE results, than for ESI-MS titration. The ESI-MS titration results showed some correlation; however, it is apparent that there are important methodological shortcomings which need to be addressed. Several factors related to the titration method, ionization efficiencies, enthalpy/entropy contributions during phase transfer, and solvent effects are discussed. This work introduces a first approach to development of a high throughput ESI-MS-based method suitable for screening potential peptide-based drug compounds that target integrins on cancerous cells. It also serves as a cautionary tale to those interested in performing similar types of analyses.

The EcoRI−DNA Complex as a Model for Investigating Protein−DNA Interactions by Atomic Force Microscopy

Atomic force microscopy (AFM) is a technique widely used to image protein-DNA complexes, and its application has now been extended to the measurements of protein-DNA binding constants and specificities. However, the spreading of the protein-DNA complexes on a flat substrate, generally mica, is required prior to AFM imaging. The influence of the surface on protein-DNA interactions is therefore an issue which needs to be addressed. For that purpose, the extensively studied EcoRI-DNA complex was investigated with the aim of providing quantitative information about the surface influence. The equilibrium binding constant of the complex was determined by AFM at both low and high ionic strengths and compared to electrophoretic mobility shift assay measurements (EMSA). In addition, the effect of the DNA length on dissociation of the protein from its specific site was analyzed. It turned out that the AFM measurements are similar to those obtained by EMSA at high ionic strengths. We then advance the idea that this effect is due to the high counterion concentration near the highly negatively charged mica surface. In addition, a dissociation of the complexes once they are adsorbed onto the surface was observed, which is weakly dependent on the ionic strength contrary to what occurs in solution. Finally, a two-step mechanism, which describes the adsorption of the EcoRI-DNA complexes on the surface, is proposed. This model could also be extended to other protein-DNA complexes.

Electrophoretic mobility shift assay on poly(ethylene glycol)‐modified glass microchips for the study of estrogen responsive element binding

The binding of estrogen receptor (ER) to estrogen response element (ERE) is essential for genomic pathways of estrogens and gel-based electrophoretic mobility shift assay (EMSA) is commonly used for analyzing ERE binding. Gel-based EMSA, however, requires the use of hazard radio isotopes and they are slow, labor-intensive and difficult to quantify. Here, we present quantitative affinity assays based on microchip electrophoresis using PEG-modified glass microchannels, which bear neutral surfaces against the adsorption of acidic DNA molecules and basic ER proteins. We first demonstrated the feasibility of the method by measuring binding constants of recombinant ERalpha and ERbeta with a consensus ERE sequence (cERE, 5'-GGTCAGAGTGACC-3') as well as with an ERE-like sequence (ERE 1576, 5'-GACCGGTCAGCGGACTCAC-3'). Changes in mobility as a function of protein-DNA molar ratios were plotted and the dissociation constants were determined based on non-linear curve fitting. The minimum amount of ER proteins required for one assay was around 0.2 ng and the run time for one chip analysis was less than 2 min. We further measured the estrogenic compound-mediated dissociation constants with recombinant ER proteins as well as with the extracted ERbeta from treated and untreated A549 bronchioloalveolar carcinoma cells. Dissociation constants determined by this method agree with the fact that agonist compounds such as 17beta-estradiol (1.70 nM), diethylstilbestrol (0.14 nM), and genistein (0.80 nM) assist ERE binding by decreasing the constants; while antagonist compounds such as testosterone (140.4 nM) and 4-hydroxytamoxifen (10.5 nM) suppress the binding by increasing the dissociation constant.

Measurement of monomolecular binding constants of neutral phenols into the β-cyclodextrin by continuous frontal analysis in capillary and microchip electrophoresis via a competitive assay

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into β-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.

Analyzing a kinetic titration series using affinity biosensors

The classical method of measuring binding constants with affinity-based biosensors involves testing several analyte concentrations over the same ligand surface and regenerating the surface between binding cycles. Here we describe an alternative approach to collecting kinetic binding data, which we call “kinetic titration.” This method involves sequentially injecting an analyte concentration series without any regeneration steps. Through a combination of simulation and experimentation, we show that this method can be as robust as the classical method of analysis. In addition, kinetic titrations can be more efficient than the conventional data collection method and allow us to fully characterize analyte binding to ligand surfaces that are difficult to regenerate.

Low Affinity Pair Size Exclusion Chromatography

Low affinity pair size exclusion chromatography (LAPSEC) is herein described as a novel approach specifically for lectin based monosaccharide separation but with the potential for wider application using weak antibody‐antigen interactions. This technique exploits weak ligand receptor interactions and the difference in molecular size between free and receptor‐bound ligand, to effect separations using a size exclusion chromatographic column. While such carrier‐based separations are achievable with high affinity interactions, the advantage of the approach described here is that the use of weak interactions also allows separation of ligand from carrier in the same column, allowing recycling of the carrier. The utility of the LAPSEC approach is shown by results obtained using Concanavalin A and Lotus Tetragonolobus lectin to separate their specific monosaccharides (D‐mannose and L‐fucose, respectively) from unbound monosaccharides. These systems have been simulated using a simple multi‐sectional equilibrium model using independent measurements of binding constants. The results show that monosaccharide separation and recovery of lectin is feasible in a single run and suggests that this approach may have potential for the selective recovery of low molecular weight products on a preparative scale. As receptors are not immobilized, resin costs are reduced, there is efficient use of affinity receptor, and separation is achieved under isocratic conditions.

Optimization of conditions for flow-through partial-filling affinity capillary electrophoresis to estimate binding constants of ligands to receptors

This work details the determination of the minimal injection time of ligand required in flow-through partial-filling affinity capillary electrophoresis (FTPFACE) to estimate binding constants of ligands to receptors. Two model systems are examined in this study: carbonic anhydrase B (CAB, EC 4.2.1.1) and arylsulfonamides, and vancomycin from Streptomyces orientalis and d-Ala- d-Ala peptides. Using CAB, a minimal injection time of 0.07 min at high pressure was determined that provided for the accurate and reproducible measurement of binding constants. In the FTPFACE technique, the capillary is first partially filled with a zone of ligand followed by a sample plug containing receptor and non-interacting standards. Upon application of a voltage the receptor and standards flow into the zone of ligand where a dynamic equilibrium is achieved between receptor and ligand. Continued electrophoresis results in the receptor and standards flowing through the domain of the ligand plug prior to detection. Analysis of the change in the relative migration time ratio (RMTR) of the receptor, relative to the non-interacting standards, as a function of the concentration of ligand, yields a value for the binding constant. In the present study, variable injection times of 4-carboxybenzenesulfonamide (CBSA) were examined to determine the minimal injection time needed to establish an equilibrium between CAB and ligand. A mathematical relationship was derived that correlated injection time and ligand concentration to the change in RMTR and comparisons made between the experimental and calculated values. Binding constants were obtained for a series of arylsulfonamide ligands and d-Ala- d-Ala terminus peptides to CAB and Van, respectively. The results support the use of FTPFACE to estimate affinity constants under variable experimental conditions.

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Binding Thermodynamics as a Tool To Investigate the Mechanisms of Drug−Receptor Interactions: Thermodynamics of Cytoplasmic Steroid/Nuclear Receptors in Comparison with Membrane Receptors

Drug-receptor binding thermodynamics has proved to be a valid tool for pharmacological and pharmaceutical characterization of molecular mechanisms of receptor-recognition phenomena. The large number of membrane receptors so far studied has led to the discovery of enthalpy-entropy compensation effects in drug-receptor binding and discrimination between agonists and antagonists by thermodynamic methods. Since a single thermodynamic study on cytoplasmic receptors was known, this paper reports on binding thermodynamics of estradiol, ORG2058, and R1881 bound to estrogen, progesterone, and androgen steroid/nuclear receptors, respectively, as determined by variable-temperature binding constant measurements. The binding at 25 degrees C appears enthalpy/entropy-driven (-53.0 </= DeltaG degrees </= -48.6, -34.5 </= DeltaH degrees </= -19.9 kJ/mol, 0.057 </= DeltaS degrees </= 0.111, and -2.4 </= DeltaC(p) degrees </= -1.7 kJ mol(-1) K(-1)) and is interpreted in terms of hydrophobic and hydrogen-bonded specific interactions. Results obtained for cytoplasmic receptors are extensively compared with those known for typical membrane receptors, in particular the adenosine A(1) receptor, to investigate the thermodynamic bases of drug-receptor binding from the most general point of view.

Affinity capillary electrophoresis analyses of protein-protein interactions in target-directed drug discovery.

Protein-protein interactions are instrumental in virtually all biological processes and their understanding will shed light on designing novel and effective drugs for therapeutic interventions targeting the pathways in which they function. Protein-protein interactions have been studied using many genetic and biochemical methods, most recently, affinity capillary electrophoresis (ACE). We used ACE as a high-throughput screening assay to establish and define binding interactions between a therapeutic target protein and chemical entities from natural product or synthetic chemical libraries. Furthermore, ACE has demonstrated its value in the measurement of binding constants, the estimation of kinetic rate constants, and the determination of the stoichiometry of protein-protein interactions. Herein, we will describe qualitatively several assay formats using ACE for detecting protein-protein interactions, and discuss their advantages and limitations.

Lipid-protein interactions studied by introduction of a tryptophan residue: the mechanosensitive channel MscL.

Trp fluorescence spectroscopy is a powerful tool to study the structures of membrane proteins and their interactions with the surrounding lipid bilayer. Many membrane proteins contain more than one Trp residue, making analysis of the fluorescence data more complex. The mechanosensitive channels MscL's of Mycobacterium tuberculosis (TbMscL) and Escherichia coli (EcMscL) contain no Trp residues. We have therefore introduced single Trp residues into the transmembrane regions of TbMscL and EcMscL to give the Trp-containing mutants F80W-TbMscL and F93W-EcMscL, respectively, which we show are highly suitable for measurements of lipid binding constants. In vivo cell viability assays in E. coli show that introduction of the Trp residues does not block function of the channels. The Trp-containing mutants have been reconstituted into lipid bilayers by mixing in cholate followed by dilution to re-form membranes. Cross-linking experiments suggest that the proteins retain their pentameric structures in phosphatidylcholines with chain lengths between C14 and C24, phosphatidylserines, and phosphatidic acid. Quenching of Trp fluorescence by brominated phospholipids suggests that the Trp residue in F80W-TbMscL is more exposed to the lipid bilayer than the Trp residue in F93W-EcMscL. Binding constants for phosphatidylcholines change with changing fatty acyl chain length, the strongest interaction for both TbMscL and EcMscL being observed with a chain of length C16, corresponding to a bilayer of hydrophobic thickness ca. 24 A, compared to a hydrophobic thickness for TbMscL of about 26 A estimated from the crystal structure. Lipid binding constants change by only a factor of 1.5 in the chain length range from C12 to C24, much less than expected from theories of hydrophobic mismatch in which the protein is treated as a rigid body. It is concluded that MscL distorts to match changes in bilayer thickness. The binding constants for dioleoylphosphatidylethanolamine for both TbMscL and EcMscL relative to those for dioleoylphosphatidylcholine are close to 1. Quenching experiments suggest a single class of binding sites for phosphatidylserine, phosphatidylglycerol, and cardiolipin on TbMscL; binding constants are greater than those for phosphatidylcholine and decrease with increasing ionic strength, suggesting that charge interactions are important in binding these anionic phospholipids. Quenching experiments suggest two classes of lipid binding sites on TbMscL for phosphatidic acid, binding of phosphatidic acid being much less dependent on ionic strength than binding of phosphatidylserine.

A model for the cooperative free energy transduction and kinetics of ATP hydrolysis by F1-ATPase.

Although the binding change mechanism of rotary catalysis by which F1-ATPase hydrolyzes ATP has been supported by equilibrium, kinetic, and structural observations, many questions concerning the function remain unanswered. Because of the importance of this enzyme, the search for a full understanding of its mechanism is a key problem in structural biology. Making use of the results of free energy simulations and experimental binding constant measurements, a model is developed for the free energy change during the hydrolysis cycle. This model makes possible the development of a kinetic scheme for ATP hydrolysis by F1-ATPase, in which the rate constants are associated with specific configurations of the beta subunits. An essential new element is that the strong binding site for ADP,Pi is shown to be the betaDP site, in contrast to the strong binding site for ATP, which is betaTP. This result provides a rationale for the rotation of the gamma subunit, which induces the cooperativity required for a tri-site binding change mechanism. The model explains a series of experimental data, including the ATP concentration dependence of the rate of hydrolysis and catalytic site occupation for both the Escherichia coli F1-ATPase (EcF1) and Thermophilic Bacillus PS3 F1-ATPase (TF1), which have different behavior.

Fluorine-NMR experiments for high-throughput screening: theoretical aspects, practical considerations, and range of applicability.

Competition ligand-based NMR screening experiments have recently been introduced to overcome most of the problems associated with traditional ligand-based NMR screening. Molecules with marginal solubility and high affinity for a given target can be easily identified in a high-throughput manner by screening chemical mixtures against the target in the presence of a weak- to medium-affinity ligand of known binding constant. While the original competition-based approaches utilized (1)H detection, significant advantages are obtained using (19)F detection. The absence of spectral overlap permits the screening of large chemical mixtures and allows for automated analysis of the spectra, even in the presence of protonated buffers, solvents, and detergents. The large chemical shift anisotropy of fluorine and the significant exchange contribution allow for the selection of a weak-affinity spy molecule, thus resulting in a lower binding affinity threshold for the identified NMR hits. The method, labeled FAXS (fluorine chemical shift anisotropy and exchange for screening) is rapid and requires only a limited amount of protein and, therefore, compares favorably with the other established non-NMR techniques used in high-throughput screening. Herein the theoretical aspects of this powerful (19)F-based approach are presented and discussed in detail. The experimental conditions together with the detection limits and binding constant measurements are investigated using human serum albumin as the target.