Biophysics in drug discovery: impact, challenges and opportunities.

Over the past 25 years, biophysical technologies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, surface plasmon resonance spectroscopy and isothermal titration calorimetry have become key components of drug discovery platforms in many pharmaceutical companies and academic laboratories. There have been great improvements in the speed, sensitivity and range of possible measurements, providing high-resolution mechanistic, kinetic, thermodynamic and structural information on compound-target interactions. This Review provides a framework to understand this evolution by describing the key biophysical methods, the information they can provide and the ways in which they can be applied at different stages of the drug discovery process. We also discuss the challenges for current technologies and future opportunities to use biophysical methods to solve drug discovery problems.

Recent trends and some applications of isothermal titration calorimetry in biotechnology.

Isothermal titration calorimeters (ITCs) are thermodynamic instruments used for the determination of enthalpy changes in any physical/chemical reaction. This can be applied in various fields of biotechnology. This review explains ITC applications, especially in bioseparation, drug development and cell metabolism. In liquid chromatography, the separation/purification of specific proteins or polypeptides in a mixture is usually achieved by varying the adsorption affinities of the different proteins/polypeptides for the adsorbent under different mobile-phase conditions and temperatures. Using ITC analysis, the binding mechanism of proteins with adsorbent solid material is derived by elucidating enthalpy and entropy changes, which offer valuable guidelines for designing experimental conditions in chromatographic separation. The binding affinity of a drug with its target is studied by deriving binding enthalpy and binding entropy. To improve the binding affinity, suitable lead compounds for a drug can be identified and their affinity tested by ITC. Recently ITC has also been used in studying cell metabolism. The heat produced by animal cells in culture can be used as a primary indicator of the kinetics of cell metabolism, which provides key information for drug bioactivity and operation parameters for process cell culture.

Applications of isothermal titration calorimetry in protein science.

During the past decade, isothermal titration calorimetry (ITC) has developed from a specialist method for understanding molecular interactions and other biological processes within cells to a more robust, widely used method. Nowadays, ITC is used to investigate all types of protein interactions, including protein-protein interactions, protein-DNA/RNA interactions, protein-small molecule interactions and enzyme kinetics; it provides a direct route to the complete thermodynamic characterization of protein interactions. This review concentrates on the new applications of ITC in protein folding and misfolding, its traditional application in protein interactions, and an overview of what can be achieved in the field of protein science using this method and what developments are likely to occur in the near future. Also, this review discusses some new developments of ITC method in protein science, such as the reverse titration of ITC and the displacement method of ITC.

Calorimetry and thermodynamics in drug design.

Modern instrumentation for calorimetry permits direct determination of enthalpy values for binding reactions and conformational transitions in biomolecules. Complete thermodynamic profiles consisting of free energy, enthalpy, and entropy may be obtained for reactions of interest in a relatively straightforward manner. Such profiles are of enormous value in drug design because they provide information about the balance of driving forces that cannot be obtained from structural or computational methods alone. This perspective shows several examples of the insight provided by thermodynamic data in drug design.

A microcalorimetric sensor for food and cosmetic analyses: l-Malic acid determination.

Enzymatic microcalorimetry has been successfully employed in the reliable determination of the l-malic acid concentration in some foods and cosmetic products. The l-malic acid concentration during the wine-making process is particularly useful in order to control the progress of the malo-lactic fermentation. Total acidity, taste and flavour characteristics of wine depend on the l-malic acid quantity still present. To point out the analytical methodology the dehydration process of l-malic acid, in the presence of Fumarase enzyme, has been used. The new method has been compared with a common spectrophotometric one. By the proposed calorimetric method the l-malic acid concentration in different types of food (white and red wines, fruits and soft beverages) has been determined. In some cosmetic products too the l-malic acid was quantified. The method outlined resulted simple, direct and reliable (good accuracy and precision), in particular it does not require any pre-treatment or clean up of the samples, save the dilution in buffer.

From biochemistry to physiology: the calorimetry connection.

This article provides guidelines for selecting optimal calorimetric instrumentation for applications in biochemistry and biophysics. Applications include determining thermodynamics of interactions in non-covalently bonded structures, and determining function through measurements of enzyme kinetics and metabolic rates. Specific examples illustrating current capabilities and methods in biological calorimetry are provided. Commercially available calorimeters are categorized by application and by instrument characteristics (isothermal or temperature-scanning, reaction vessel volume, heat rate detection limit, fixed or removable reaction vessels, etc.). Advantages and limitations of commercially available calorimeters are listed for each application in biochemistry, biophysics, and physiology.

Advances in the analysis of isothermal titration calorimetry data for ligand-DNA interactions.

Isothermal titration calorimetry (ITC) is a well established technique for the study of biological interactions. The strength of ITC is that it directly measures enthalpy changes associated with interactions. Experiments can also yield binding isotherms allowing quantification of equilibrium binding constants, hence an almost complete thermodynamic profile can be established. Principles and application of ITC have been well documented over recent years, experimentally the technique is simple to use and in ideal scenarios data analysis is trivial. However, ITC experiments can be designed such that previously inaccessible parameters can be evaluated. We outline some of these advances, including (1) exploiting different experimental conditions; (2) low affinity systems; (3) high affinity systems and displacement assays. In addition we ask the question: What if data cannot be fit using the fitting functions incorporated in the data-analysis software that came with your ITC? Examples where such data might be generated include systems following non 1:n binding patterns and systems where binding is coupled to other events such as ligand dissociation. Models dealing with such data are now appearing in literature and we summarise examples relevant for the study of ligand-DNA interactions.