Isothermal titration calorimetry: application to structure-based drug design

The road to market for drug compounds is a treacherous one, generally involving a huge temporal and financial investment. The role of structure-based drug design or lead optimisation ranges wildly in importance in different pharmaceutical companies. The adoption of these aids to provide routes to high affinity ligands has not received widespread acceptance. This is based on a number of factors, from the perceived failings of such methods, to the belief that rapid screening of compound libraries alone is the most effective way to discover drugs.

The panacea of being able to take a computer generated representation of the structure of a target site of a given biomolecule and rationally design an high affinity inhibiting compound has proved seemingly unreachable for three major reasons: (1) current capabilities in computing; (2) the requirement for atomic resolution structural detail; and (3) determination of how structural features can be related to the thermodynamics of interactions. It is the last of these points that this review seeks to address. In particular the use of isothermal titration calorimetry is discussed in the light of the accumulation of accurate thermodynamic data and examples are given where this has been applied to understand the structural aspects of formation of drug–biomolecular complexes.     

Flow sorption calorimetry, a powerful tool to investigate the acid-base character of organic polymer surfaces

Flow sorption calorimetry (FSC) can be a useful tool to estimate the Lewis acid-base behaviour of the surface of organic polymers with less than 1 m²/g surface area. Polyethylene powders grafted with different amounts of acrylic acid have been chosen as samples. The surface of the powders was investigated by means of X-ray photoelectron spectroscopy (XPS) at first and than brought into contact with the strong electron donor triethylamine in a special FSC measuring system. The heat of the displacement isotherm together with the surface excess isotherm of triethylamine adsorbed onto the powders was determined. The results obtained with FSC and XPS were well correlated and gave a comprehensive picture of the acidic surface character of modified polyethylene. Received: 19 September 1996 / Revised: 22 January 1997 / Accepted: 26 January 1997     

Isothermal titration calorimetry (ITC) method to study drug/ion exchanger interaction

An isothermal titration calorimetry (ITC) method to measure the heat effects evolving from the binding between cation exchanger Amberlite ® IRP 69 and the cationic drug substances propranolol hydrochloride (PROP), metoprolol tartarate (METO), acebutolol hydrochloride (ACEB) and chlorpheniramine maleate (CPR) has been developed. The method gives repeatable results with an error about 5% for the beta-blockers PROP, METO and ACEB, and about 10% for the antihistamine CPR. The calculation of the thermodynamic parameters enthalpy change (ΔH _bind) and Gibbs free energy change (ΔG _bind) show significant differences between the different drug substances.     

Isothermal titration calorimetry of Ni(II) binding to histidine and to N-2-aminoethylglycine

Isothermal titration calorimetry (ITC) is used to study the complexation thermodynamics of Ni(II) with histidine (His) and with N-2-aminoethylglycine (EDMA). The titrations were performed in HEPES and Tris buffers at various ionic strengths and pH values around 8. The results show the influence of the experimental conditions on the shape and fitting parameters of the calorimetric curves. For the studied systems, the main reaction is concomitant with a number of side reactions which contribute to the global energy measured. From the calorimetric data measured, the formation constants for the species NiHEPES⁺, Tris⁺His⁻, TrisNiHis⁺ and [Ni(EDMA)₂OH]⁻ have been evaluated for the first time and the values obtained properly validated.     

A new tool for an old job: Using fixed cell scanning calorimetry to investigate dilute aqueous solutions

The development of fixed twin cell temperature scanning calorimeters has enabled the more efficient determination of heat capacities of dilute aqueous solutions with a precision comparable to that of the Picker flow heat capacity calorimeters developed nearly 40 years ago. Experiments require less than 0.5 cm³ of solution, and results can be obtained routinely over the temperature range (278 to 395) K at pressures up to a few bars. Multiple scanning of samples by both increasing and decreasing temperature allows assessment of instrument drift, solute stability, and reproducibility of results. Chemical calibration is essential to take full advantage of the precision and sensitivity of the calorimeters. The calorimetric output is a direct measure of the difference in heat capacity per unit volume of a solution and of a reference liquid, usually water. Thus, densities of the solution and reference liquid are needed to transform the results into heat capacities per unit mass of solution. Examples of solutes that have been investigated include a variety of inorganic and organic compounds that dissolve to give simple ionic or neutral species, or that produce complexes or species that exist in equilibrium distributions that can change as the temperature is scanned. Appropriate selection of the results from experiments on combinations of solutes allows calculation of standard state (zero concentration) thermodynamic quantities for chemical processes and reactions over the ranges of temperature scanned at the solution compositions investigated. Results for a few specific systems are presented and discussed for some representative classes of solutes that have been investigated in our laboratory since 1998.     

Contact angle measurements as a tool to investigate the filler-matrix interactions in polyurethane-clay nanocomposites from blocked prepolymer

Optically transparent polyurethane-clay nanocomposite films were prepared by dispersing 5 wt% of various commercial organo-clays in a mixture of cycloaliphatic amines used as chain extender-cross-linker of a blocked prepolymer. For the first time, vibration-induced equilibrium contact angle measurements were successfully employed to rank the selected organo-clays accordingly to their hydrophobicity order. Polymer-clay intercalation degree in the nanocomposites, as assessed from X-ray diffraction, was strongly correlated to the water-clay equilibrium contact angle. Moreover, as the clay intercalation degree increased, a decrease of the cross-linking degree of the polyurethane matrix occured.Uniaxial tensile tests under quasi-static and impact conditions, and isothermal thermogravimetric analysis were performed on both unfilled polyurethane matrix and nanocomposites. Secant tensile modulus, tensile energy to break, and thermal lifetime showed a non monotonic trend with a maximum as a function of the intercalation degree. This behaviour is discussed considering that two concomitant and contrasting effects develop as the polymer-clay intercalation degree increases: a positive improvement of the filler matrix interactions, and a negative reduction of the matrix cross-linking degree.     

Isothermal titration calorimetry and dynamic light scattering studies of interactions between gemini surfactants of different structure and Pluronic block copolymers

The interactions between triblock copolymers of poly(ethylene oxide) and poly(propylene oxide), P103 and F108, EO(n)PO(m)EO(n), m=56 and n=17 and 132, respectively, and m-s-m type gemini surfactants, m=8, 10, 12, and 18, and s = 3, 6, 12, and 16, have been studied in aqueous solution using isothermal titration calorimetry and dynamic light scattering techniques. The enthalpograms of F108 as a function of surfactant concentration show one broad peak at polymer concentrations C(p) < or = 0.50 wt%, below the cmc of the copolymer at 25 degrees C. It is attributed to interactions between the surfactant and the triblock copolymer monomer. DLS results show hydrodynamic radii (R(h)) initially consistent with copolymer monomers that change to values consistent with gemini surfactant micelles as the surfactant concentration is increased. In P103 solutions at C(p) > or = 0.05 wt%, two peaks appear in the enthalpograms, and they are attributed to the interactions between the gemini surfactant and the micelle or monomer forms of the copolymer. An origin-based nonlinear fitting program was employed to deconvolute the two peaks and to obtain estimates of peak properties. An estimate of the fraction of copolymer in aggregated form was also obtained. The enthalpy change due to interactions between the surfactants and P103 aggregates is very large compared to values obtained for traditional surfactants. This suggests that extensive reorganization of copolymer aggregates and surrounding solvent occurs during the interaction. DLS results for the P103 systems containing C(p) > or = 0.05% show evidence of very large aggregates in solution, likely P103 micelle clusters. The transitions observed in the hydrodynamic radii are consistent with a breakdown of micelle clusters with addition of gemini surfactant, followed by mixed micelle formation and/or deaggregation into monomer P103. This is followed by interactions similar to those typically observed in surfactant-nonionic polymer systems. Mechanisms for the interaction and the observed structural changes are discussed.     

Isothermal calorimetry as a tool for estimating resistance of wild oat (Avena fatua L.) to aryloxyphenoxypropionate herbicides

The application of isothermal calorimetry for the early detection of the resistance of wild oat to fenoxaprop¹ and diclofop² was investigated. In the first test, three leaf tillers were sprayed with field doses of fenoxaprop or diclofop. For resistant biotypes, the rate of heat flow after 48 h was similar to that in control plants. In susceptible biotypes, fenoxaprop significantly reduced and diclofop significantly increased the rate of heat flow. In the second test, 3-day-old seedlings were put into calorimetric ampoules on filter paper moistured with herbicide solution (152% and 40% of the field dose for fenoxaprop and diclofop, respectively). Rate of heat flow was measured for 72 h, however, differences were already visible in the first hours of germination on each herbicide. Rate of heat flow for seedlings resistant to both herbicides was higher than for susceptible ones. The most evident differences between susceptible and resistant biotypes were noticed after 10-20 h and 25-40 h (of the seedlings’ growth) on fenoxaprop and diclofop, respectively, when a sharp increase of rate of heat flow was observed. In conclusion, calorimetry may be used as a rapid test for the detection of the resistance of wild oat biotypes to fenoxaprop and diclofop.     

Isothermal titration calorimetry studies on the binding of DNA bases and PNA base monomers to gold nanoparticles

An isothermal titration calorimetric (ITC) investigation of the interaction of DNA bases and PNA base monomers with gold nanoparticles is described revealing a binding sequence in the order C > G > A > T. Direct measurement of the strength of interaction of ligands with nanogold by ITC has important implications in surface modification strategies for biomedical, catalysis, and nanoarchitecture applications.     

Cation-pi interactions as a tool to enhance the power of a chiral auxiliary during asymmetric photoreactions within zeolites

Owing to the existence of cation-pi interactions, aryl chiral auxiliaries perform far better than alkyl chiral auxiliaries during asymmetric photoreaction.     

Differential scanning calorimetry as an analytical tool in the study of drug-cyclodextrin interactions

The interactions of trimethoprim, sulphadiazine and sulphamethoxazole with natural (a- b-, g- ) and amorphous (RAMEB) or crystalline (DIMEB) methylated b-cyclodextrins were investigated both in aqueous solution (using phase-solubility analysis) and in the solid state (using DSC supported by X-ray analysis). In particular, DSC studies enabled determination of the relative degree of crystallinity of each drug in its physical and ground mixtures with the different cyclodextrins on the basis of the variation of its heat of fusion in comparison with that of the pure drug. In all cases, the host cavity size was a prevalent factor for the inclusion complexation in liquid state. On the contrary, it had a negligible effect on solid-state interactions in terms of drug amorphization. DIMEB and RAMEB exhibited similar performances in aqueous solution, showing that the presence of methyl-groups improved the complexing and solubilizing properties of b-cyclodextrin. However, DSC studies revealed that RAMEB was clearly more active in performing solid-state interactions, i.e. drug amorphization, and as stabilizing agent for the amorphous state brought forth. This revised version was published online in July 2006 with corrections to the Cover Date.     

The electron swarm method as a tool to investigate multi-body electron attachment processes

The electron swarm method has been used for the investigation of multi-body thermal electron attachment process. Ethylene, carbon dioxide and their mixtures were used as carrier gases. The rate constant for the reaction of thermal electrons with SF₆ has been measured and found to be (2.5±0.2) × 10^-7 cm³·mol^-1·s^-1. The mechanism and kinetics of thermal electron capture by N₂O was investigated in the mixtures with C₂H₄ and CO₂. A mechanism involving neutral van der Waals molecules (N₂O·CO₂) has been proposed and the rate constants calculated to be 1.9 × 10^-10 cm³·mol^-1·s^-1 for the formation of (N₂O·CO₂) and 1.8 × 10^-30 cm⁶·mol^-2·s^-1 for the three-body reaction with CO₂ as a stabilizing agent.     

Strategies for assessing proton linkage to bimolecular interactions by global analysis of isothermal titration calorimetry data

Isothermal titration calorimetry (ITC) is a traditional and powerful method for studying the linkage of ligand binding to proton uptake or release. The theoretical framework has been developed for more than two decades and numerous applications have appeared. In the current work, we explored strategic aspects of experimental design. To this end, we simulated families of ITC data sets that embed different strategies with regard to the number of experiments, range of experimental pH, buffer ionization enthalpy, and temperature. We then re-analyzed the families of data sets in the context of global analysis, employing a proton linkage binding model implemented in the global data analysis platform SEDPHAT, and examined the information content of all data sets by a detailed statistical error analysis of the parameter estimates. In particular, we studied the impact of different assumptions about the knowledge of the exact concentrations of the components, which in practice presents an experimental limitation for many systems. For example, the uncertainty in concentration may reflect imperfectly known extinction coefficients and stock concentrations or may account for different extents of partial inactivation when working with proteins at different pH values. Our results show that the global analysis can yield reliable estimates of the thermodynamic parameters for intrinsic binding and protonation, and that in the context of the global analysis the exact molecular component concentrations may not be required. Additionally, a comparison of data from different experimental strategies illustrates the benefit of conducting experiments at a range of temperatures.     

Scanning force microscopy as a tool to investigate the properties of polyglycerol ester foams

Foamed products are a popular class of food products. The mechanism of stabilization of the air bubbles is often only partially understood. The current study aims at better understanding the stabilization of air-water interfaces through the low molecular weight surfactant polyglycerol ester (PGE). We chose PGE films as an exemplary case for a non-equilibrium situation at an air-water interface--a situation that requires the development of new experimental techniques. Several different film preparation and transfer methods onto solid substrates have been tested. The films were then investigated by scanning force microscopy, and structural artifacts associated to the sample preparation were identified and discussed. In addition to the study of Langmuir monolayers and Gibbs adsorption layers, we have proposed a new approach to investigate the skins of foam bubbles. We thereby were able to determine that PGE indeed covers bubbles by a multilayer structure and that the pH plays a role in the structuring of the films. We show that a combination of different film preparation methods allows us to get an insight into the aggregation behavior of PGE at the air-water interface and thereby better understand the stabilization mechanism of this particular surfactant.     

The quartz crystal microbalance: a new tool for the investigation of the bioadhesion of diatoms to surfaces of differing surface energies

Diatoms are a major component of the biofoul layer found on modern low-surface-energy, 'foul release' coatings. While diatoms adhere more strongly to hydrophobic, as opposed to hydrophilic, surfaces, surprisingly little is known of the chemical composition of their adhesives. Even less is known about the underlying processes that characterize the interaction between the adhesive and a given surface, including those of differing wettability. Using the quartz crystal microbalance with dissipation monitoring (QCM-D), we examined differences in the viscoelastic properties of the extracellular adhesives produced by the marine diatoms Amphora coffeaeformis Cleve and Craspedostauros australis Cox interacting with surfaces of differing wettability; 11-mercaptoundecanoic acid (MUA) that is hydrophilic and 1-undecanethiol (UDT) that is hydrophobic. While the overall delta f/delta D ratios were slightly different, the trends were the same for both diatom species, with the layer secreted upon UDT to be more viscoelastic and far more consistent over several experiments, compared to that on MUA which was less viscoelastic and demonstrated far more variability between experiments. While the nature of the parameter shifts for C. australis were the same for both surfaces, A. coffeaeformis cells settling upon UDT illustrated significant positive f and D shifts during the initial stages of cell settlement and adhesion to the surface. Further experiments revealed the parameter shifts to occur only during the initial adhesion of cells upon the pristine virgin UDT surface. The mechanism behind these parameter responses was isolated to the actin-myosin/adhesion complex (AC), using the myosin inhibitor 2,3-butanedione 2-monoxime (BDM) to remove the cells ability to 'pull' on adhesive strands emanating from the cell raphe. The observations made herein have revealed that adhesives secreted by fouling diatoms differ significantly in their interaction with surfaces depending on their wettability, as well as illustrating the unique mechanics behind the adhesion of A. coffeaeformis upon hydrophobic surfaces, a mechanism that may contribute significantly to the cells success in colonizing hydrophobic surfaces.     

NMR spectroscopy as a tool to investigate the degradation of aromatic compounds by a Pseudomonas putida strain

Pseudomonas putida strain KT2442, harbouring the pWW0 TOL plasmid, was grown with a number of different homologous aromatic acids as carbon sources. Small samples of liquid culture supernatant were collected and directly analysed by 2D NMR spectroscopy. In all cases similar compounds with olefinic signals were observed to accumulate. To elucidate the structures of these compounds, 2D NMR experiments with 500 and 600 MHz spectrometers equipped with a CryoProbe (Bruker BioSpin) were performed on samples obtained from a culture growing on 4‐methylbenzoate and, for ¹³C spectroscopy, on ¹³C‐labelled 4‐methylbenzoate. In all cases a 1,2‐dihydroxycyclohexa‐3,5‐diene‐carboxylate derivative was identified. The use of this technique helped us to identify easily some metabolites that were released into the solution by bacteria and to follow their secretion as a function of time. The high sensitivity of the present approach allowed a clear and rapid acquisition of spectra, notwithstanding the low concentration of the compounds. The benefits of introducing the use of NMR cryoprobes to perform metabolic pathway studies is demonstrated. Copyright © 2003 John Wiley & Sons, Ltd.     

Quartz crystal microbalance with dissipation coupled to on-chip MALDI-ToF mass spectrometry as a tool for characterising proteinaceous conditioning films on functionalised surfaces

Proteinaceous conditioning films (pCFs) are thought to play a key role in microbial adhesion, leading to the fouling of technical and biomedical devices and biofilm formation, which in turn causes material damage or persistent infections, respectively. However, little is definitively known about the process of surface conditioning via proteins. Herein, we demonstrate the potential of quartz crystal microbalance with dissipation coupled to MALDI-ToF mass spectrometry (QCM-D-MALDI) to investigate protein adsorption on different surfaces, enabling both the monitoring of CF formation and the determination of the molecular composition of CFs. After running QCM-D experiments, a subsequent tryptic on chip digestion step allows the identification of the proteins deposited on the sensor chip surface via MALDI-ToF mass spectrometry. Prominent blood plasma proteins, i.e., human serum albumin (HSA), fibrinogen (FG) and fibronectin (FN), were used. Chemically well defined sensor surfaces were prepared, among others, via self-assembled monolayer (SAM) technology. In cases where protein adsorption was observed by QCM-D, the adsorbed proteins were clearly detected and identified using MALDI-ToF/MS for both single-protein solutions of HSA, FG and FN as well as for protein mixtures. However, for equimolar protein mixtures on TiO₂ surfaces, only signals attributed to FG and FN were observed in the mass spectra. No signals indicating the presence of HSA could be detected. This finding leads to the assumption that only FG and FN attach to the TiO₂ sensor surface under the given experimental conditions.