Kinetic and thermodynamic assessment of binding of serotonin transporter inhibitors

Several serotonin reuptake inhibitors are in clinical use for treatment of depression and anxiety disorders. However, to date, reported pharmacological differentiation of these ligands has focused mainly on their equilibrium binding affinities for the serotonin transporter. This study takes a new look at antidepressant binding modes using radioligand binding assays with [(3)H]S-citalopram to determine equilibrium and kinetic rate constants across multiple temperatures. The observed dissociation rate constants at 26 degrees C fall into a narrow range for all molecules. Conversely, association rate constants generally decreased with increasing equilibrium binding affinities. Consistent with this, the measured activation energy for S-citalopram association was relatively large (19.5 kcal . mol(-1)), suggesting conformational change upon ligand binding. For most of the drugs, including citalopram, the enthalpy (DeltaH(O)) and entropy (-TDeltaS(O)) contributions to reaction energetics were determined by van't Hoff analyses to be roughly equivalent (25-75% DeltaG(O)) and to correlate (positively for enthalpy) with the polar surface area of the drug. However, the binding of the drug fluvoxamine was predominantly entropically driven. When these data are considered in the context of the physicochemical properties of these ligands, two distinct binding modes can be proposed. The citalopram-type binding mode probably uses a polar binding pocket that allows charged or polar interactions between ligand and receptor with comparatively small loss in enthalpy due to dehydration. The fluvoxamine-type binding mode is fueled by energy released upon burying hydrophobic ligand moieties into a binding pocket that is flexible enough to suffer minimal loss in entropy from conformational constraint.     

Interactions of the catharanthus (Vinca) alkaloids with tubulin and microtubules.

The dimeric Vinca alkaloids represent a group of important anti-tumor compounds whose intracellular target is tubulin, the protein monomer of microtubules. In this review data on the binding of these drugs to tubulin and microtubules in vitro are examined. The binding to tubulin is linked to a protein self-association reaction described by Na and Timasheff (1986a) as a ligand-induced plus ligand-mediated isodesmic self-association reaction. The simplest model which fits the binding data is one in which there is one intrinsic site which is linked to the self-association process. Effects of solution variables on the binding and self-association explain the wide variation of reported apparent binding constants for Vinca alkaloids to tubulin. The Vinca drugs also bind to microtubules via a low number of sites at the ends of microtubules with apparent high affinity and which are involved in the inhibition of tubulin dimer addition to the microtubule ends, and to sites along the microtubule wall with apparent low affinity which are involved in the disruption of the microtubules into spiraled protofilaments. This review also compares available binding data for different natural and semi-synthetic Vinca alkaloids.     

Thermodynamic analysis of the temperature dependence of the dissociation constant of naloxone at opioid delta receptors in the mouse isolated vas deferens.

Dissociation constants (KB) for naloxone inhibition of the actions of DPDPE in the mouse isolated vas deferens preparation (inhibition of electrically induced twitch) were determined at five temperatures ranging between 25 and 40 degrees C. The values of KB tended to increase with temperature over the range examined, indicating that the affinity of naloxone for the opioid delta receptor is an inverse function of temperature. Using these data, the thermodynamic quantities delta G zero' (change in free energy), delta H zero' (change in enthalpy) and delta S zero' (change in entropy) were calculated from a van't Hoff plot of in (KB) against 1/T. The thermodynamic quantities determined in this study in vivo (delta G zero' = -10.59 kcal mol-1, delta H zero' = -15.73 kcal mol-1 and delta S zero' = -0.0168 kcal mol-1 zero K-1) are consistent with data reported from radioligand binding studies in vitro and suggest that the interaction between naloxone and the opioid delta receptor in the mouse isolated vas deferens is enthalpy driven. These data represent the first evaluation of the thermodynamics of opioid antagonist/receptor interaction in a physiological assay.     

The pharmacological basis of receptor binding.

Radiolabeled ligands can be used to characterize recognition sites of natural receptors. Verification of a receptor-specific binding of a radioligand requires a series of in vitro approaches, as saturation, competition, and kinetic (association and dissociation) experiments. Success of ligand binding depends on may factors involving the radio-pharmaceutical used as the radioligand, methods for separating bound and free ligand, proper use of the non-specific binding marker, and proper analysis of the binding data. A major theoretical constraint is the very short time available for separating bound an free radioligand, since a considerable fraction of radioligand can dissociate from the receptor binding sites during this process, in particular if the receptor affinity of the radioligand is low. Saturable, steady-state binding data can be analyzed utilizing standard binding isotherms and non-linear regression analysis as well as Scatchard plots. Competition experiments with unlabeled drugs are the most important in determining pharmacology of binding. Each method is subject to potential artifacts. Estimation of association is straightforward in order to assess the time necessary to achieve binding equilibrium.     

Measurement of thermodynamic parameters for norepinephrine contraction of isolated rabbit thoracic aorta

The thermodynamic quantities of change in free energy (delta G degree'), change in enthalpy (delta H degree') and change in entropy (delta S degree') were determined for the interaction of norepinephrine with the alpha-1 adrenoceptor of vascular smooth muscle. Specifically, a standard isolated rabbit thoracic-aorta preparation was used to examine the effect of temperature on norepinephrine-induced isometric tension development. Dissociation constants (KA) for norepinephrine were determined at several temperatures over the range 25-40 degrees C from equiactive concentrations obtained before (A) and after (A') partial irreversible receptor blockade by phenoxybenzamine, plotted as 1/A against 1/A' (KA = (slope-1)/intercept). The values of KA increased with temperature over the range 25-40 degrees C, indicating that the affinity of norepinephrine for the alpha-1 adrenoceptor is an inverse function of temperature over this range. From these results, the thermodynamic quantities delta H degree' and delta S degree' were determined from a van't Hoff plot of In (KA) against 1/T. The relative magnitudes of the change in enthalpy (delta H degree' = -25.58 kcal mol-1) and the change in entropy (delta S degree' = -0.052 kcal mol-1 deg-1) suggest that the reaction between norepinephrine and the alpha-1 adrenoceptor (delta G degree' = -9.15 kcal mol-1) is enthalpy driven, which is consistent with radioligand binding studies of other adrenoceptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of pheromone‐binding protein specificity

Many pheromones have very low water solubility, posing experimental difficulties for quantitative binding measurements. A new method is presented for determining thermodynamically valid dissociation constants for ligands binding to pheromone‐binding proteins, using β‐cyclodextrin as a solubilizer and transfer agent. The method is applied to LUSH, a Drosophila odorant‐binding protein that binds the pheromone 11‐cis vaccenyl acetate (cVA). Refolding of LUSH expressed in Escherichia coli was assessed by measuring N‐phenyl‐1‐naphthylamine (NPN) binding and Förster resonance energy transfer between LUSH tryptophan 123 (W123) and NPN. Binding of cVA was measured from quenching of W123 fluorescence as a function of cVA concentration. The equilibrium constant for transfer of cVA between β‐cyclodextrin and LUSH was determined from a linked equilibria model. This constant, multiplied by the β‐cyclodextrin‐cVA dissociation constant, gives the LUSH‐cVA dissociation constant: ～100 nM. It was also found that other ligands quench W123 fluorescence. The LUSH‐ligand dissociation constants were determined to be ～200 nM for the silk moth pheromone bombykol and ～90 nM for methyl oleate. The results indicate that the ligand‐binding cavity of LUSH can accommodate a variety ligands with strong binding interactions. Implications of this for the Laughlin, Ha, Jones and Smith model of pheromone reception are discussed.     

Fluorescent Assay for Estimating the Binding of Erythromycin Derivatives to Ribosomes

A fluorescent erythromycin derivative was used to determine the binding of erythromycin derivatives to ribosomes. The assay is rapid, sensitive, and convenient. Because measurements are made in solution, they represent equilibrium conditions, unlike filter binding assays which perturb the equilibrium. Results correlate well with measurements made by other techniques.     

Characteristics of high-affinity folate binding in human leukocytes

High-affinity binding of [3H]folate in leukocytes from normal subjects was studied in equilibrium dialysis experiments (pH 7.4, 37 degrees C). Binding displayed positive cooperativity, and the binding affinity increased with decreasing concentration of the binding protein. Both phenomena could be interpreted in terms of ligand binding to a polymerizing system where the affinity of ligand for the oligomer is greater than its affinity for the polymer prevailing at higher concentrations of the binding protein.     

Competition in drug binding and … the race to equilibrium

Binding kinetics has become a popular topic in pharmacology due to its potential contribution to the selectivity and duration of drug action. Yet, the overall kinetic aspects of complex binding mechanisms are still merely described in terms of elaborate algebraic equations. Interestingly, it has been recommended some 10 years ago to examine such mechanisms in terms of binding fluxes instead of the conventional rate constants. Alike the velocity of product formation in enzymology, those fluxes refer to the velocity by which one target species converts into another one. Novel binding flux-based approaches are utilized to get a better visual insight into the “competition” between two drugs/ligands for a single target as well as between induced fit- and conformational selection pathways for a single ligand within a thermodynamic cycle. The present data were obtained by differential equation-based simulations. Early on, the ligand-binding steps “race” to equilibrium (i.e., when their forward and reverse fluxes are equal) at their individual pace. The overall/global equilibrium is only reached later on. For the competition association assays, this parting might produce a transient “overshoot” of one of the bound target species. A similar overshoot may also show up within a thermodynamic cycle and, at first glance, suggest that the induced fit pathway dominates. Yet, present findings show that under certain circumstances, it could rather be the other way round. Novel binding flux-based approaches offer visually attractive insights into crucial aspects of “complex” binding mechanisms under non-equilibrium conditions.     

Dynamics and partitioning of spin-labeled stearates into the lipid domain of stratum corneum.

The EPR spectra of the positional isomers n-doxyl stearic acids (n-DSA), with n=5, 12 and 16, and 5-doxyl methyl stearate (5-DMS) structured in the lipid domain of intact stratum corneum (SC), are characterized by the thermodynamic equilibrium of two distinct spectral components provided by two different motional states of the spin-labeled chains. A two-component model used in the EPR spectra simulations provided the relative populations of the components, allowing for the calculation of the thermodynamic profile. Based on a detailed investigation, the more motionally restricted population of spin labels (component 1) is found to arise when the spin label is hydrogen-bonded to the polar surfaces of the membranes, while the less motionally restricted population (component 2) is generated by spin labels nonhydrogen-bonded and more deeply inserted in the hydrophobic core. The 5-DSA is bound tightly to the polar surfaces (DeltaG(o)2 --> 1=-1.75 kcal/mol and DeltaH(o)2 --> 1=-13.8 kcal/mol), whereas the more lipophilic 5-DMS has a major spin population stabilized in the hydrophobic core (DeltaG(o)2 --> 10.57 kcal/mol and DeltaH(o)2 --> 1=-9.1 kcal/mol). Upon lipid-depleting SC increases the interactions of the probe with the polar surfaces, thereby decreasing its rotational diffusion. In contrast, the treatment of SC with oleic acid, a permeation enhancer, drastically increases the mobility of the spin labels, particularly that of component 1, and the thermodynamic equilibrium shifts towards the formation of component 2. A mechanism for water permeation in SC is also proposed.     

Dynamic equilibrium between protein S and C4b binding protein is important for accurate determination of free protein S antigen.

Protein S in circulation is in a dynamic equilibrium with C4b binding protein (C4bBP), thus affecting the measurement of free protein S antigen. We addressed the issue of overestimation of the free protein S concentration with current immunoassays due to the dynamic equilibrium and propose a new method for its accurate determination. Our assay system was tested at different reaction temperatures using purified free protein S, protein S-C4bBP complexes, plasma samples, and a commercially available free protein S assay kit. At a reaction temperature of 37 degrees C, the free protein S fraction increased from 0.5 ng/ml (at 4 degrees C) to 7.8 ng/ml, and from 4.5 ng/ml (at 4 degrees C) to 56 ng/ml when the concentration of the assayed protein S-C4bBP complexes was 20 ng/ml and 200 ng/ml, respectively. In plasma samples, free protein S levels were approximately 0.8 microg/ml and 6 pg/ml higher at 25 degrees C and 37 degrees C, respectively compared to measurements at 4 degrees C. Measurements of free protein S in plasma using a commercially available assay kit were approximately 0.6 microg/ml higher at 25 degrees C than measurements performed at 4 degrees C. Dynamic equilibrium between protein S and C4bBP affects the measurement of free protein S antigen. Measurement of free protein S antigen should be performed under conditions where protein S is not dissociated from protein S-C4bBP complexes, as exemplified by assay at low temperature (4 degrees C).     

Problems in the measurement of iron binding capacity in serum

The magnesium carbonate method for measurement of the total iron binding capacity and unsaturated iron binding capacity was studied to disclose causes of error and improve techniques. Failure to regulate the pH of reaction mixtures at all stages of the procedure and inadequate amounts of protein in the specimen each caused a significant error in measurements of both the “total” and “unsaturated” iron binding capacities. An exchange of unbound radioiron in the iron saturating solution with transferrin bound iron caused an increase in UIBC values. The use of undiluted serum specimens, regulation of the pH of iron saturating solutions and the addition of sodium barbital buffer to serum before the addition of iron saturating solution seemed to improve the measurements.     

Determination of free concentration of piroxicam and naproxen in plasma. The influence of experimental conditions in equilibrium dialysis

An equilibrium dialysis method was established in order to investigate possible relationships between free drug concentrations of piroxicam and naproxen and clinical events. Therefore the influence of variations in pH, phosphate concentration and sodium azide concentration of the dialysis buffer on the free concentrations of piroxicam and naproxen was investigated. Piroxicam was found to have a pH-dependent protein binding. Therefore a good control of pH during the dialysis process is necessary. This has been achieved by increasing the buffer capacity of the dialysis buffer, by adding an antibacterial agent to the dialysis buffer and by cleansing the dialysis cells with 70% ethanol before use to prevent bacterial growth. Addition of 0.03% sodium azide as an antibacterial agent and the use of a 0.09 mol/l phosphate buffer gave good pH control. A method to correct for deviations of pH in measurements of free concentrations of piroxicam by a simple mathematical correction has been found. As naproxen was found to have a protein binding independent of pH, a pH-correction is not necessary for this drug. Standardized conditions in determination of protein binding of drugs by equilibrium dialysis are important, as composition of the dialysis buffer and pH of plasma compartment at equilibrium may influence the free concentration measurements. Comparisons of data from experiments using different methods are therefore difficult; the importance of pH-control is stressed. With the methods used in the present investigation, equilibrium dialysis in connection with HPLC, the coefficients of variation for piroxicam and naproxen free concentrations are 5.5% and 7.4%, respectively.     

Practical applications of radioimmunoassay theory. A simple procedure yielding linear calibration curves.

The fundamental equation describing radioimmunoassays under equilibrium conditions has been recast into a "working equation" in a form more directly applicable to the requirements of the analytical laboratory. Plotting total counts over counts bound vs. ligand concentration, which is conveniently linear over most of its course, is shown readily to yield quantitative data relative to binding site concentration and the equilibrium constant and to provide a means for deriving apparent labeled ligand concentration. Such data are helpful in establishing optimum assay conditions and can serve a continuing quality-control function. The working equation also characterizes the binder and tracer reagents used in the assay. The determination of working-equation parameters has been illustrated for the vitamin B-12 assay. Data are presented for seven different assay procedures, involving more than 600 calibration curves and 100 different lots of binding agent and tracer reagent, showing a consistently high correlation coefficient (r greater than 0.990), between ligand concentration and the response variable.     

Topological investigation of the reaction mechanism of glycerol carbonate decomposition by bond evolution theory

The reaction mechanisms of the decomposition of glycerol carbonate have been investigated at the density functional theory level within the bond evolution theory. The four reaction pathways yield to 3-hydroxypropanal (TS1), glycidol (TS2a and TS2b), and 4-methylene-1,3-dioxolan-2-one (TS3). The study reveals non-concerted processes with the same number (four) of structural stability domains for each reaction pathway. For the two decarboxylation mechanisms, the two first steps are similar. They correspond to the cleavage of two single CO bonds to the detriment of the increased population of the lone pairs of two O atoms. These are followed, along TS1, by the transformation of a CO single bond into a double bond together with a proton transfer to create a CH bond. For TS2a and TS2b, the last step is a cyclization by CO bond formation. For the TS3 pathway, the first stage consists in the cleavage of a CH bond and the transfer of its electron population to both a proton and a C atom, the second step corresponds to the formation of an OH bond, and the last one describes the formation of a CC double bond. Moreover, the analysis of the energies, enthalpies, and free enthalpies of reaction and of activation leads to the conclusion that 3-hydroxypropanal is both the thermodynamic and kinetic product, independent of the method of calculation.

ELFs of glycerol carbonate and of its kinetic and thermodynamic decomposition product, 3-hydroxypropanal (+CO₂).     

RECEPTORS ON IMMUNOCOMPETENT CELLS : IV. DIRECT MEASUREMENT OF AVIDITY OF CELL RECEPTORS AND COOPERATIVE BINDING OF MULTIVALENT LIGANDS

The interaction of antigen with specific, cell-associated receptors was measured in thermodynamic terms. The binding of ¹²⁵I-labeled 2,4-dinitrophenyl guinea pig albumin (DNP₁₆GPA-¹²⁵I) to lymphocytes from guinea pigs immunized to DNP₁₆GPA is a temperature-dependent, reversible process. Measurement of association and dissociation rates of antigen-receptor complexes permits calculation of antigen-cell binding constants. These may also be calculated by equilibrium-binding techniques. Although differences in the constants calculated in these two ways exist, a clear increase in avidity of cell receptor for antigen occurs in the course of the immune response. This change in receptor avidity provides evidence that the time-dependent change in affinity of serum antibody (maturation) indeed has a cellular basis. The magnitude of the equilibrium constant is, in part, due to binding of more than one DNP group per molecule of antigen. Thus, multivalent ligands bind more effectively to cell receptors than univalent or paucivalent ligands when measured by the number of antigen molecules bound, the dissociation rate of antigen-receptor complexes, and in the relative capacity to inhibit a standard multivalent ligand (DNP₁₆GPA-¹²⁵I) from binding.     

Tracer kinetic model of blood-brain barrier transport of plasma protein-bound ligands. Empiric testing of the free hormone hypothesis.

Previous studies have shown that the fraction of hormone or drug that is plasma protein bound is readily available for transport through the brain endothelial wall, i.e., the blood-brain barrier (BBB). To test whether these observations are reconcilable with the free-hormone hypothesis, a tracer-kinetic model is used in the present investigations to analyze in vivo initial extraction data on BBB transport of protein-bound steroid hormones (dihydrotestosterone, testosterone, estradiol, and corticosterone), thyroid hormones (triiodothyronine), and lipophilic amine drugs (propranolol). The plasma proteins used are bovine albumin and human orosomucoid. Transport data was fit to a modification of the Kety-Renkin-Crone equation of capillary physiology; the modified equation incorporates the principles of both capillary physiology and plasma protein-ligand mass action binding relationships. In most cases, the experimental data is best fit to the model equation when the apparent in vivo dissociation constant, KDa, of the ligand protein binding reaction increases to values that are 5- to 50-fold greater than the in vitro dissociation constant, KD. This result indicates that the rate of ligand dissociation from the plasma protein is accelerated in the capillary bed relative to the in vitro situation. It is hypothesized that the major factor leading to the rapid transport in vivo of protein-bound ligands into tissues such as brain is an endothelial-induced decrease in the affinity of the plasma protein for the ligand. Under these conditions, the amount of plasma ligand available for tissue clearance in vivo parallels the protein-bound fraction, not the free hormone.     

Immunoreactivity of the Rho(D) antigen in cytoskeleton-free vesicles

Skeleton-free microvesicles derived from D-positive red cells immunospecifically bind anti-D. Anti-D saturation binding yielded an equilibrium constant value (K = 1-2 X 10(8) I mole-1) similar to intact red cells. The vesicles contained band 3, the major sialoglycoproteins and, at high protein loads, low-molecular-weight polypeptides. Anti-D binding was referenced to total protein, phospholipid, and band 3 content to determine whether there was nonrandom segregation of the D antigen into vesicles. Selective segregation of the D antigen into vesicles could not be demonstrated unequivocally based on the methods and the assumptions of this study. The results, however, indicate that D reactivity does not require a membrane skeletal association.     

Polychromatic analysis: new applications of an old technique.

As a means of correcting for many potential interferences, polychromatic analysis offers an effective alternative to either sample pretreatment or separate blank determinations. Because of the differing spectral characteristics of each interfering species, and the nature of these interferences (static vs. kinetic), the application of polychromatic analysis must be optimized for each analytical procedure to minimize all residual errors. Overall assay precision can be maximized by proper use of flagging procedure to minimize all residual errors. Overall assay precision can be maximized by proper use of flagging procedures (also based on polychromatic analysis). By using a second wavelength measurement, the analyst can also verify the accuracy of an analytical measurement in selected situations. These applications of polychromatic analysis are a departure from conventional analytical procedures in allowing for correction of various interferences in the actual reaction mixture, without making assumptions about the recovery of analyte during pretreatment or the equivalent absorptivity of interferent in both blank and assay solutions.     

Combined quantum mechanics/molecular mechanics (QM/MM) methods to understand the charge density distribution of estrogens in the active site of estrogen receptors

The ligand binding to protein and host–guest interactions are ubiquitous for molecular recognition. In drug design, the ligand binding to the active site of proteins is influenced by the charge density distribution and the electrostatic interactions of ligands and the nearby amino acids of the protein. The charge density analyses of ligand–protein complexes need accurate positions of hydrogen atoms and their valence electron distribution and the fine structure of proteins. Such information cannot be obtained from the conventional protein X-ray crystallography analysis in the resolution range of 1.5 to 3.5 Å. This can be realized from QM/MM based structure and charge density analysis of estrogens with the estrogen receptor. The charge density properties such as electron density, Laplacian of electron density and electrostatic properties of estrogens in the presence of active site amino acid residues have been determined and compared with the isolated estrogen molecules from theory and experimental. The present study reveals the chemical bonding nature of estrogen molecules and the strength of the intermolecular interactions in the active site of estrogen receptor, and also the importance of π⋯π interactions between the estrogens and Phe404 amino acid residue and protonation state of His524 amino acid residue have been identified using electrostatic potential maps. The difference in the electrostatic potential map of estrogens displays the hormone dependent actions of estrogen receptor. This method is very helpful to derive the charge density distribution of macromolecules to understand their biological recognition and interactions.

The ligand binding to protein and host–guest interactions are ubiquitous for molecular recognition.