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.

Physical activity assessment in the general population; instrumental methods and new technologies

The objective measurement of human movement and the quantification of energy expenditure due to physical activity is an identified need in both research and the clinical setting. Validated and well-defined reference methods (double labelled water, direct calorimetry, indirect calorimetry) are expensive and mostly limited to the laboratory setting. Therefore, in the last years, several objective measurement devices have been developed which are appropriate for field studies and clinical settings. There is no gold standard among them, as all have limitations. Pedometers are small, non-expensive, count the steps taken and give information on total physical activity, but not about physical activity patterns and behaviour. Accelerometers are expensive, save information about frequency and intensity of physical activity, but not about type of physical activity. Both pedometers and accelerometers only save information about lower body movement, but reliability about the estimation of energy expenditure is limited. Heart rate monitoring relates intensity to energy expenditure, but gives no information about physical activity. GPS watches are portable, relatively inexpensive, non-invasive and provide distance, speed, and elevation with exact time and location, but are maybe limited for the assessment of brief higher speed movement and energy expenditure. Combined motion sensors combine accelerometry with the measurement of physiological variables and share advantages of single devices and are more precise. Manufacturer software which applies activity-specific algorithms for the calculation of energy expenditure can affect energy expenditure results. Most of the devices estimate energy expenditure more accurately at light to moderate intensities; underestimation increases at very light and higher intensity activities (AU)

La medición objetiva del movimiento humano y la cuantificación del gasto energético debido a la actividad física es una necesidad identificada tanto en investigación como en clínica. Los métodos de referencia validados y bien definidos (el agua doblemente marcada, la calorimetría directa, la calorimetría indirecta) son caros y prácticamente se limitan a la investigación en el laboratorio. Por lo tanto, en los últimos años, se han desarrollado diferentes dispositivos de medición objetiva que son apropiados para los estudios de campo y clínicos. No hay ningún estándar de oro entre ellos, ya que todos tienen limitaciones. Los podómetros son ligeros, poco costosos, cuentan los pasos y aportan información sobre la actividad física total, pero no sobre el comportamiento y los patrones de actividad física. Los acelerómetros son caros, aportan información sobre patrón, frecuencia e intensidad de la actividad física, pero no sobre el tipo de actividad física. Los podómetros y acelerómetros únicamente recogen información sobre el movimiento del movimiento corporal, pero la validez en la estimación del gasto energético es limitada. La monitorización de la frecuencia cardíaca relaciona intensidad del ejercicio con gasto de energía, pero no aporta información sobre la actividad física. Los dispositivos GPS son portátiles, relativamente asequibles, no invasivos y recogen distancia, velocidad y elevación con hora y lugar exactos, pero quizás estén limitados para la evaluación de movimientos cortos de alta intensidad y elevado gasto energético. Los dispositivos de última generación combinan acelerometría con la medición de variables fisiológicas, comparten las ventajas de los dispositivos individuales y son más precisos. Para el cálculo del gasto energético se aplican algoritmos específicos de la actividad incluidos en el software del fabricante que pueden afectar a los resultados. La mayoría de los dispositivos estiman con mayor precisión el gasto energético a intensidades ligeras y moderadas, pero subestiman el gasto a intensidades muy ligeras y de mayor intensidad (AU)

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.

Determinación de la estabilidad de Kollicoat MAE 30D mediante medidas de calorimetría diferencial de barrido / Determination of the stability of Kollicoat MAE 30D by means of differential scanning calorimetry

Se estudia la estabilidad de Kollicoat MAE 30D mediante determinaciones calorimétricas y microscópicas. Se estudian los efectos que producen pH, temperatura y agitación sobre las propiedades fisicoquímicas de las partículas de este látex comercial. Se comprueba que el factor que más influencia ejerce es el pH, siendo máxima la estabilidad a pH ácidos, y modificándose con un decrecimiento de la misma, a partir de pH 6. Se determina la forma y tamaño de partícula mediante medidas microfotográficas de SEM, realizando el recuento de 200 partículas de una muestra del látex previamente desecado. Se comprueba que la dispersión acuosa de Kollicoat MAE 30 D a su pH natural(2.5), da lugar a una población de partículas con un diámetro medio de 1.56 nm (AU)

The stability of Kollico at MAE 30 D was studied with differential scanning calorimetric, and scanning electron microscopy. We investigated the effects of pH, temperature and shaking on physicochemical properties of the latex particles. Stability of the polymer as most strongly affected by pH; hence, we conclude that the stability of this latex reaches a maximum at acid pH values, whereas stability is lost to pH 6.The particle shape and the mean diameter were determined by means of SEM microphotographs on 200 particles in a sample of dry latex. The aqueous Kollico at MAE 30 D dispersion, at its natural pH (2.5), presented one population of particles with a mean diameter of 1.56 nm (AU)

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.

Ritmo de sudoración como mejor indicador de aclimatación al calor / Sweat rate as best indicator of heat aclimation

Objetivo: Determinar cual de los tres índices de aclimatación comúnmente utilizados (temperatura timpánica, ritmo de sudoración, o frecuencia cardiaca) es el más sensible y reproducible. Material y métodos: Siete jóvenes estudiantes universitarios (28+/- asños y 4,2+/-0,6 L/min VO2max) pedalearon a una intensidad del 60% del VO2max durante 60 minutos por 7 días (2 días de descanso máximo) en una cámara climática a 34,4+/-0,3ºC y 27,7+/-0,1% de humedad relativa, con 0,2 m/s de velocidad de viento. Este proceso de aclimatación se repitió 3 veces (A1, A2 y A3) espacidas por 3 meses para estudiar la reproducibilidad de las respuestas. Resultados: Tras 3 días de aclimatación el ritmo de sudoración (RS) subió por encima del ritmo del día 1 en todas las aclimataciones (p<0.05) pero no alcanzó un estado estable tras 7 días. La reproducibilidad entre aclimataciones fue alta para el ritmo de sudoración (R1=0.90), y frecuencia cardiaca final (R1=0.71). Conclusiones: El ritmo de sudoración es el índice más sensible y reproducible de aclimatación y recomendamos su uso en vez de frecuencia cardíaca o temperatura timpánica. Aunque los efectos de una semana de aclimatación al calor se pierden en 3 mese (día 1 de A1, A2 y A3 son similares) las respuestas son altamente reproducibles. Por último, confirmamos datos anteriores que más de 7 días son necesarios para alcanzar la máxima adaptación en ritmo de sudoración y frecuencia cardíaca

Purpose: to determine which of three commonly used indexed to evaluate acclimation (tympanic temperature, sweat rate, or heart rat is the most sensitive and reliable). Material and methods: seven young University students (28+/-8 years and 4.2+/-0.6 L/min VO2max) pedalled at 60% del VO2max for 60 minutes during 7 days (maximum of 2 rest days in between) in a climatic chamber set at 34.4+/-0.3ºC and 27.7+/-0.1% of relative humidity with 0.2 m/s of wind speed. This acclimation protocol was repeated in 3 occasions () A1, A2 y A3) separated by 3 months to study reliability. Results: After 3 days of acclimation sweat rate was higher than on day 1 in all (p<0.05) although a steady-state was not achieved at day 7. Tympanic temperature at the end of exercise was unchanged with no effect of days of acclimation. Heart rate at the end of exercise was lower than the day 1 after 5-6 days in all acclimations (p<0.05) without reaching a steady-state. There were a high reliability in sweat rate (R1=0.90) and heart rate (R1=0.84) but moderate for tympanic temperature (R1=0.71). Conclusions: Sweat rate is the most sensitive indication of acclimatation and we recommended its use over heart rate or tympanic temperature. Although the effects 1 week acclimation are lost within 3 months (day 1 of A1, A2, and A3 are similar) the responses are highly reliable. Finally, we confirm previous reports that indicates that indicate that over 7 days are needed to reach a steady-state response in sweat rate and heart rate

Monitorización no invasiva de la PaCO2 en pacientes críticos con ventilación mecánica / Noninvasive monitorization of PaCO2 in critically ill ventilated patients

Objetivo. Determinar la presión arterial de dióxido de carbono, en pacientes con soporte ventilatorio completo, mediante dos técnicas de monitorización no invasivas, la calorimetría indirecta (eliminación de CO2) y la capniografía (presión teleespiratoria de CO2). Comparar sus resultados frente a los obtenidos mediante la extracción de gasometrías arteriales. Métodos. Veinte pacientes en ventilación mecánica, sometidos a un cambio en el volumen ventilatorio, fueron monitorizados con un computador metabólico (Deltatrac) y un capniógrafo. Se determinaron las siguientes variables antes y después del cambio ventilatorio: eliminación de CO2 (VeCO2), consumo de oxígeno, PETCO2 y PaCO2 (gasometría arterial). Se calcularon los nuevos niveles de PaCO2 mediante calorimetría indirecta utilizando PaCO2-final = PaCO2-basal × VeCO2-basal/VeCO2-1 minuto, y mediante capniografía, utilizando PaCO2-final = PETCO2-final + Grad (PaCO2PETCO2). Resultados. Los niveles de PaCO2 obtenidos mediante calorimetría indirecta fueron de 37 (DE 5,2) mmHg y mediante la PETCO2 de 35,4 (8,3) mmHg; tras la extracción de gases arteriales los niveles fueron de 37,1 (6,4) mmHg y 37,2 (6,7) mmHg, respectivamente. La correlación entre la PaCO2 calculada y la medida mediante gasometrías arteriales fue de r = 0,93 (p < 0,001) para la calorimetría indirecta y de r = 0,55 (p < 0,05) para la PETCO2. En el análisis de concordancia de ambas técnicas frente a la gasometría arterial los límites de acuerdo fueron más estrechos para la calorimetría indirecta (-5,11/5,31 mmHg frente a -12,35/16,07 mmHg, margen de confianza del 95 por ciento). Conclusiones. En nuestro estudio se confirma la escasa utilidad de la PETCO2 para la determinación de los cambios sufridos por la PaCO2 en pacientes críticos en los que se han variado los parámetros ventilatorios (AU)