Biomimetic characterization of drug–protein binding process by quasi-two-dimensional sensing techniques

The interaction of neuroactive kynurenic acid (KYNA) with human serum albumin (HSA) was studied from both thermodynamic and structural point of view. This protein-drug interaction has high relevance in neuroscience, because the serum albumin-based KYNA-containing composites may act as a potential drug delivery system. Reversible binding of KYNA on HSA has been investigated under physiological conditions at various temperatures. The overall results of isothermal titration calorimetry (ITC), fluorescence spectroscopy and two-dimensional surface plasmon resonance (SPR) techniques showed similar trend for the temperature dependence of the estimated association constant (KA) values. Moreover, the enthalpy- (ΔH⁰), entropy- (ΔS⁰) and heat capacity changes (ΔCp) of KYNA-HSA complex formation were also calculated and interpreted according to the integrated van't Hoff equation. The results indicate low-affinity complex formation between the protein and the drug molecule, with two independent binding sites. The presence of the drug in the protein caused significant structural change with an α-helix content decrease from 69.7% to 51.2% proved by circular dichroism (CD) measurements. Theoretical calculations also support the experimentally suggested two independent binding sites theory stabilized by salt bridges and aromatic π-π stacking in the proposed binding positions.

Target and appiled model: The studied system is specifically relevant in the neuroscience. Glutamate is the main excitatory neurotransmitter in the CNS. Kynurenic acid (KYNA) is the only known endogenous antagonist of ionotropic glutamate-receptors (GluR), and an increasing evidence suggests its neuroprotective capacity. At a higher, micromolar concentration range KYNA is an inhibitor of the GluR, while a few years ago it has been demonstrated to have facilitatory effect on GluRs in a lower, nanomolar concentration level. Understanding the exact molecular mechanism of the action of KYNA on GluRs might promote future drug development for therapeutic management of neurological disorders Kynurenic acid (KYNA) Introduction: The development of optical sensor or microfluidic devices as well as the computing background provide an increasing opportunity to characterize and monitor chemical and biological quantities. Sensor techniques that allow monitoring chemical parameters of molecular or colloidal systems in liquid environments are needed for the experimental assessment of receptor-ligand type interactions for modern pharmaceutical developments. This work presents a detailed description of a commonly used and low-cost evaluation process that is simply based on the nonlinear fitting of concentration-, time-and temperature-dependent surface plasmon resonance (SPR) sensorgrams in order to provide kinetic and thermodynamic characterization of a biologically relevant interaction. Regardless of type, this evaluation proceeding is suitable to manage the time-dependent sensor signals of other quasi two-dimensional sensor techniques, like quartz crystal microbalance (QCM) or optical waveguide lightmode spectroscopy (OWLS).

Analytical methods based on the surface plasmon resonance (SPR) phenomena have become widely used in the last decades as a result of the rapid development of the electronics and information technology background. In the early stages of the development of this technique, the method was primarily characterized by the study of compounds bound to the surface of the sensor, but nowadays it is also possible to study complex systems such as the interaction of lipid bilayers that model cell membranes or liposomes formed from phospholipids and proteins. Among the quasi two-dimensional sensor techniques, the unlabelled, quantitative, real-time and temperature-dependent characterizations of receptor-ligand type interactions can be carried out by using SPR technique at the interface between the receptor-coated sensor surface and the ligand solution. Internationally, the SPR is an extremely widely used sensor technology, but according to the publications at Hungarian Institutes there are only a few papers which interpret the results of the detailed characterization of molecular interactions. During my research work, one of the main goal was to determine the individual cross sectional area of several proteins, amino acid, and di- and tripeptides. In order to confirm the applicability of this technique the experimentally determined cross sectional areas have been compared with analogous and structural data provided by quartz crystal microbalance (QCM) method and small angle X-ray scattering investigation, respectively. Beyond the determination of the quantitative relationship between the surface-immobilized macromolecules and their ligands, the definition of the rate and equilibrium constants of the binding process by developing an appropriate data processing and evaluation process is also an integral part of my dissertation. Among others, according to the above-mentioned evaluation process, the study of the pH-dependent binding process of ibuprofen (IBU) onto the bovine serum albumin (BSA)-functionalized sensor surface has been carried out. Based on the fitting of the registered sensorgrams with kinetic models a spreadsheet-based software solution has been developed which resulted in the determination of the association and dissociation kinetic constants. In order to verify the binding parameters isotherm titration calorimetry (ITC) studies have also been performed. The thermodynamic state functions determined by studying the temperature-dependence of the equilibrium constant can reinforce or refute the validity of a supposed reaction mechanism. During the preparation of my dissertation, the implementation and evaluation of temperature-dependent SPR measurements formed the backbone of my research work and these results have been presented in detail. The aim of the temperature-dependent measurements was the determination of the thermodynamic parameters of the interaction between AMPA receptor model peptides synthesized by solid-phase Fmoc synthesis (University of Szeged, Department of Medical Chemistry) and kynurenic acid (KYNA) and their synthetic derivatives. Moreover, comparative studies have also been carried out where the model receptor polypeptides were substituted by serum proteins and lysozyme (LYZ). In view of the thermodynamic parameters the validity of the assumed binding mechanism was confirmed by independent ITC measurements, where the compounds were available in enough quantities.

This work presents a detailed description of a commonly used and low-cost evaluation process that is simply based on the nonlinear fitting of concentration-, time- and temperature-dependent surface plasmon resonance (SPR) sensorgrams in order to provide kinetic and thermodynamic characterization of a biologically relevant interaction. Regardless of type, this evaluation proceeding is maximally suitable to manage the time-dependent sensor signals of other quasi two-dimensional sensor techniques, like quartz crystal microbalance (QCM) or optical waveguide lightmode spectroscopy (OWLS). Based on the hypothetic reaction schemes, the SPR signals have been fitted by the analytical solutions of the rate equations using optional spreadsheet software. Statistical tests of the goodness of nonlinear fits confirm the application of a reversible, pseudo first order kinetic model, in this way the apparent (k obs ), the association (k a ) and the dissociation (k d ) constants as well as the equilibrium constants (K A ) and the Gibbs free energy change (ΔG) have been calculated at six different temperatures. The enthalpy (ΔH ⁰ ), entropy (ΔS ⁰ ) and heat capacity changes (ΔC p ) have also been estimated using nonlinear regression analysis. The sign and values of the state functions approve the presence of a salt bridge between a model polypeptide fragment of human glutamate receptor (ANYT) and kynurenic acid (KYNA) which was confirmed by molecular docking calculations as well.

Investigation of receptor – ligand interactions play a determinant role in molecular life science. Detailed quantitative, kinetic and thermodynamic characterization of these biomolecular interactions may decisively contribute to the modern pharmaceutical developments. Surface plasmon resonance spectroscopy is capable of real-time monitoring of these interactions without use of labels via immobilization one of the binding partners (protein or small biomolecule) onto the sensor surface. Understanding the exact molecular mechanism of the action of kynurenic acid on Human Glutamate Receptor (GluR1) peptide fragments might promote future drug development for the therapeutic management of neurological disorders. In our study reversible bonded amount of kynurenic acid have been measured on different peptide-modified gold surface at different temperatures under physiological conditions 1. Besides the experimental characterization of the receptor-ligand systems the underlying molecular mechanism can be investigated using the methods of computational molecular modelling. The binding mode and structural properties of a peptide or protein adsorbed on a surface can be elucidated using molecular dynamics (MD) simulations2. The main object of our SPR experiments is to provide important parameters (quantitative data of bounded amount of drug molecule on different protein-covered solid support; kinetic (rate of association and dissociation) and thermodynamic (ΔG, ΔH, ΔS) data for better understanding of the mechanism of protein-drug molecule bindings. Acknowledgement This work was supported by the Hungarian Research Fund OTKA K116323. References 1. Csapó, E. et al. Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments. Colloids Surf. B. Biointerfaces 123, 924–9 (2014). 2. Csapó, E. et al. Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments. Part 2: Interaction of GluR1270-300 with KYNA. Colloids Surf. B. Biointerfaces 133, 66–72 (2015).

Introduction: Detailed kinetic and thermodynamic characterization of physiological relevant receptor-ligand interactions provides several important data on the mechanisms of the drug binding and release process. Surface plasmon resonance (SPR) spectroscopy is capable of real-time monitoring of these interactions without use of labels via immobilization one of the binding partners (endogen bioligands such as human serum albumin, transferrin, DNA etc.) onto the sensor surface. Ruthenium complexes have emerged among the most promising alternatives to platinum based compounds (cisplatin, carboplatin and oxaliplatin) which are undoubtedly successful anticancer drugs. Ruthenium compounds such as the organometallic moiety (pentamethylcyclopentadienyl)rhodium(III) (RhCp*) and its complexes have different physico-chemical and pharmacokinetic behavior compared to the platinum drugs and they have different mechanism of action, in this way they are the subject of extended drug discovery efforts. Recognition the exact molecular mechanism of the action of ruthenium compounds on endogen bioligand (human serum albumin: HSA) might promote future metal-based anticancer drug development. Results and Discussion: In our study reversible bonded amount of metal complexes or ligands have been measured on different HSA-modified gold surface at different temperatures under physiological conditions. The main object of our SPR experiments was to provide quantitative kinetic (rate of association and dissociation) and thermodynamic data for better understanding of the mechanism of the assumed protein-drug molecule bindings. The extracted sensorgrams were fitted via nonlinear parameter estimation method by using pseudo first order kinetic model. Based on the concentration dependence of the estimated observed rate constants the real rate constants as well as the equilibrium constants and the Gibbs free energy change were calculated at different temperatures. The temperature dependent enthalpy- and entropy change for binding interaction were obtained by nonlinear regression analysis with integrated van’t Hoff equation. Methods: The sensorgrams were measured via a wavelength modulated, temperature-controlled and two-channel SPR imaging apparatus (developed at the Institute of Photonics and Electronics, Prague, Czech Republic) and recorded by the SPR UP 1.1.11.3 (2014 IPE AS CR) control software. The evaluation of extracted data was carried out in an own development spreadsheet environment and nonlinear parameter estimation based software proceeding.

Quantitative detection of desired target chemicals in a sensitive and selective manner is extremely important to protect human health and environment. Nature has been a complete foundation of inspiration for the planning of sensitive and selective sensor techniques. For targeted and controlled drug delivery systems the study of the interactions between the carrier and a drug molecule play a determinant role in pharmaceutical developments. Exhaustive kinetic and thermodynamic characterization of proteinligand and membrane-ligand interactions provide several important data on the mechanisms of the drug binding and release process. Surface plasmon resonance (SPR) spectroscopy is capable of real-time monitoring technique of these interactions via immobilization or building one of the binding partners (protein, peptide or molecular bilayer architecture) onto the sensor surface. Understanding the exact molecular mechanism of the action of drugs on proteins, receptor- and membrane modeless might promote future drug development for the therapeutic management of neurological disorders and cancer therapies. The main object of our SPR experiments was to provide quantitative kinetic (rate of association and dissociation) and thermodynamic data for better understanding of the nature and mechanism of receptor-drug and carrier-drug bindings [1]. The SPR sensorgrams were fitted via nonlinear parameter estimation method by using pseudo first order kinetic model [2]. Based on the concentration dependence of the estimated observed rate constants the real rate constants as well as the equilibrium constants (KA) and the Gibbs free energy (ΔG) change were calculated at different temperatures. The temperature dependent enthalpy- and (ΔH) entropy (ΔS) change for binding interaction were obtained by nonlinear regression analysis with integrated van’t Hoff equation [3]. Comparison of the results of investigated systems give those kind of evidences such as the formation of a salt bridge between kynurenic acid and the positively charged residues of the receptor model polypeptide (Arg, Lys) at pH 7.4, which earlier has been suggested by molecular docking calculations as well. Existing of specific binding affinity and way between and AMPA receptor model peptide fragments. In the case of bovine serum albumin based core-shell carrier the release mechanism of the drug can be influenced by the protein concentration in the inner phase, not only by forming shells around the protein core. Acknowledgements The research was supported by the National Research, Development and Innovation Office-NKFIH through the project “Synthesis, structural and thermodynamic characterization of nanohybrid systems at solid-liquid interfaces” K116323 and GINOP-2.3.2-15-2016-00038 and GINOP-2.3.2-15-2016 00034. Ádám Juhász gratefully acknowledges the financial support of UNKP-17-3 research fellowship. [1] Edit Csapó, Ádám Juhász, Noémi Varga, et al. Colloids and Surfaces A, 2016, 504, 471. [2] Ádám Juhász, Edit Csapó, Ditta Anita Ungor, et al. The Journal of Physical Chemistry B, 2016, 120, 7844. [3] Ádám Juhász, Edit Csapó, László Vécsei, et al. Periodica Polytechnica Chemical Engineering, 2017, 61, 3.

For protein-based controlled drug delivery systems the study of the interactions between the carrier and a drug molecule play a determinant role in pharmaceutical developments. Detailed kinetic and thermodynamic characterization of protein-ligand interactions provide several important data on the mechanisms of the drug binding and release process. Surface plasmon resonance (SPR) spectroscopy is capable of real-time monitoring of these interactions without use of labels via immobilization one of the binding partners (protein or peptide) onto the sensor surface. Understanding the exact molecular mechanism of the action of kynurenic acid on receptor fragments and serum albumins might promote future drug development for the therapeutic management of neurological disorders. In our study reversible bonded amount of kynurenic acid have been measured on different peptide-modified gold surface at different temperatures under physiological conditions 1,2. The main object of our SPR experiments was to provide quantitative kinetic (rate of association and dissociation) and thermodynamic data for better understanding of the mechanism of protein-drug molecule bindings. The SPR sensorgrams were fitted via nonlinear parameter estimation method by using pseudo first order kinetic model. Based on the concentration dependence of the estimated observed rate constants the real rate constants as well as the equilibrium constants (KA) and the Gibbs free energy (ΔG) change were calculated at different temperatures. The temperature dependent enthalpy- and (ΔH) entropy (ΔS) change for binding interaction were obtained by nonlinear regression analysis with integrated van’t Hoff equation. Comparison of the results of investigated systems gives an evidence of the existing of specific binding affinity and way between kynurenic acid and earliest investigated AMPA receptor peptide fragments. References 1. Juhász, Á. et al. Kinetic and Thermodynamic Evaluation of Kynurenic Acid Binding to GluR1 270–300 Polypeptide by Surface Plasmon Resonance Experiments. The Journal of Physical Chemistry B 120, 7844–7850 (2016). 2. Juhász, Á., Csapó, E., Vécsei, L. & Dékány, I. Modelling and Characterization of the Sorption of Kynurenic Acid on Protein Surfaces. Periodica Polytechnica Chemical Engineering 61, 3–9 (2017).

Bimetallic silver–gold nanoparticles (NPs) with different structures were prepared. Namely, silver–gold alloy and core(Ag)–shell(Au) type nanoparticles were synthesized at various Ag/Au ratios using only sodium citrate as reductant without any stabilizers. The synthesized NPs were characterized by different structure analysis methods. The unique optical properties of these NPs were studied by UV–vis spectroscopy. For Ag/Au alloy NPs the λmax values are linearly tunable from ∼408 nm to 525 nm depending on the composition. The plasmon band of core–shell NPs can be shifted to higher wavelengths by depositing a gold shell with increasing thickness. The HRTEM images clearly confirm the two different structures of the NPs. We found that the size of alloy nanoparticles decrease from d ∼15 nm to d ∼8 nm with the increase in gold content. In contrast, core(Ag)–shell(Au) type NPs with progressively increasing size (d = 13–16 nm) along with separately formed smaller gold NPs were formed.

This paper presents a surface plasmon resonance (SPR) spectroscopy-based evaluation process which provides information on kinetic and thermodynamic aspects of the interactions between proteins and a drug molecule. Reversible binding of kynurenic acid (KYNA) on human (HSA) and bovine (BSA) serum albumin-modified gold sensor surface has been investigated under physiological conditions at various temperatures. The SPR sensorgrams were fitted via nonlinear parameter estimation method by using pseudo first order kinetic model. Based on the concentration dependence of the estimated observed rate constants (kobs) the association (ka) and dissociation (kd) constants as well as the equilibrium constants (KA) and the Gibbs free energy (ΔG0) change were calculated at different (10-35 C°) temperatures. Furthermore, the enthalpy (ΔH0), entropy (ΔS0) and heat capacity changes (ΔCp) of KYNA-protein complex formation were also calculated. © 2017, Budapest University of Technology and Economics. All rights resereved.

This work clearly demonstrates an evaluation process that is easily performed and is simply based on the fitting of temperature-dependent surface plasmon resonance (SPR) sensorgrams in order to provide detailed thermodynamic characterization of biologically relevant interactions. The reversible binding of kynurenic acid (KYNA) on human glutamate receptor (GluR1) polypeptide (GluR1270-300)-modified gold surface has been studied at various temperatures under physiological conditions by two-dimensional SPR experiments. The registered sensorgrams were fitted by using different kinetic models without application of any commercial softwares. Assuming that the association of GluR1270-300-KYNA complex is first order in both reactants the association (ka) and dissociation (kd) constants as well as the equilibrium constants (KA) and the Gibbs free energy change (ΔG0) were given at 10, 20, 30 and 40 °C. Moreover, the enthalpy (ΔH0 = -27.91 kJ mol-1), entropy (ΔS0 = -60.33 J mol-1 K-1) and heat capacity changes (ΔCp = -1.28 kJ mol-1 K-1) of the model receptor-ligand system were also calculated with a spreadsheet program. Negative values of ΔG0 and ΔH0 indicate the exothermic formation of a stable GluR1270-300-KYNA complex, since the |ΔH|>|TΔS| relation suggests an enthalpy-driven binding process. The negative ΔH0 and ΔS0 strongly support the formation of a salt bridge between KYNA and the positively charged residues of the polypeptide (Arg, Lys) at pH 7.4 confirmed by molecular docking calculations as well.

The interactions between bovine serum albumin (BSA) and ibuprofen (IBU) were investigated at pH 3.0 and pH 7.4 by several two-(2D) and three-(3D) dimensional techniques to provide quantitative, kinetic and thermodynamic data on the BSA-IBU binding. Based on the results, the preparation of BSA-IBU composite nanoparticles (NPs) were successfully carried out for controlled drug release. The high resolution transmission electron microscopy (HRTEM), dynamic light scattering (DLS) and small angle x-ray scattering (SAXS) studies confirm the formation of nearly monodisperse NPs with daverage = 10–13 nm depending on the protein concentrations and IBU contents. The kinetics of pH-induced drug release was studied by a vertical diffusion cell at pH 7.4 at 25 °C. The pH-dependent changes in the secondary structure of BSA were proven by SAXS, DLS and surface plasmon resonance (SPR) investigations. Depending on the protein conformations, the SPR results suggest that the bonded amounts of the drug molecule are 1239 mg IBU/g BSA and 174 mg IBU/g BSA at acidic and neutral pH, respectively. Besides quantification of the interactions, the rate of association (ka) and dissociation (kd), the KA and KD standard equilibrium constants and the binding free energy (ΔG°) were also calculated on the basic of SPR measurements. The ΔG° = − 21.5 ± 0.2 kJ mol−1 obtained by SPR in 2D system is in good agreement with the ΔG° = − 17.38 ± 0.54 kJ mol−1 determined by isotherm titration calorimetry (ITC) in solution (3D).