Direct reading of Microcal Origin OPJ files with AFFINImeter

Direct reading of Microcal Origin OPJ files with AFFINImeter

The direct reading  Microcal Origin OPJ files has been recently implemented in AFFINImeter. Initially, only OPJ files containing a single datasheet were allowed. Our code has just been modified to make possible the reading of OPJ files with multiple datasheets. Additionally the uploaded OPJ files are now being filtered to permit just the selection of datasheets suitable for analysis with AFFINImeter (i.e. datasheets of ITC isotherms). Some minor improvements that optimize this process have also been implemented.
Soon you will also be able to directly upload Microcal Origin itc files. In collaboration with our scientific advisor Prof. Philippe Dumas, from the University of Strasbourg, we are currently implementing a procedure to automate the integration of the raw ITC data, including removal of noise and baseline correction.

How to perform a Global Fitting Analysis?

The Global fitting of multiple isotherms is one of the advanced tools that AFFINImeter offers to facilitate the analysis and interpretation of isothermal titration experiments and to expand the range of applications of this technique.

The following video tutorial describes the global fitting of three isotherms of a displacement assay describing, a receptor interacting with a tight ligand, with a weak ligand, or with both ligands simultaneously, in a competitive experiment where the ligands are mixed in the syringe of the ITC equipment.

 

If you want to know more about global fittings with AFFINImeter you can also download the case of use “Global Analysis in ITC Displacement Titrations with AFFINImeter” that describes a Displacement Titration Assay to determine the thermodynamics of HIV-protease with indinavir, a high-affinity binder, and with acetyl-pepstatin, a weaker ligand.

ITC displacement titrations offer an attractive alternative to standard assays when working with ultra-high or ultra-low- affinity interacting systems. The method requires the fitting of at least two isotherms that share various adjustable parameters. The case study exemplifies the potential advantages of using AFFINImeter in ITC displacement assays. The software offers unique advanced tools that enhance the robustness of the method and makes it more versatile, facilitating the acquisition of reliable thermodynamic data from ultra-high of ultra-low affinity systems. Thus, it opens a door for new applications of the displacement assay.

 

 

 

Competitive Binding Assay

Competition assays

Competitive binding assays where two (or more) ligands bind to the same receptor have become common experiments in many research areas, from basic investigations to innovation in the pharmaceutical industry. These assays can be done in different formats, i.e. through a displacement assay where ligand “L1” is displaced by ligand “L2” from a preformed complex “L1-receptor” or via titration of a receptor solution with a mixture of L1 +L2. Either way, the competitive binding assay provides rich thermodynamic and structural information of the various binding events taking place during the course of the experiment. Thus, Isothermal Titration Calorimetry (ITC) competition assays performed in a displacement format have been revealed as an efficient tool for the quantitative analysis of very high- / low- interactions, with application in the field of fragment based drug screening (ref).

Analysis of competition experiments with AFFINImeter

The versatility of the experimental setup in AFFINImeter-ITC permits the analysis of ITC competition experiments in its various formats. As an illustration, the following lines describe the analysis of an ITC isotherm resulting from a competitive experiment where a solution of a receptor in titrated with mixture of two competing ligands.

AFFINImeter contains a series of examples with which users can practice and learn the overall process of data fitting: from equipment and data uploading to fitting model design and data fitting.

In this post we will review an example of a competitive model fitting model used to analyze the experiment data of two ligands in the syringe competing for binding to the same receptor.

Practical case: competitive binding model

The AFFINImeter example “competitive binding model” illustrates an ITC experiment where two ligands, “A” and “B” compete with each other for binding to the receptor “M”.

Drawing of a competitive binding model
Competitive Binding model scheme

This situation corresponds to a binding model consisting of three free species (A, B and M) two binding equilibria representing the interaction of M with A and M with B

The model was designed with the “reaction builder” and stored in “models”.The equipment used is decribed and stored in “equipments”. The dataseries is uploaded and stored in “dataseries”. When the dataseries is uploaded, the user has to complete the information relative to the equipment used and the species concentration. In this particular case (where there is a competitor “B”).

How to fit a project with AFFINImeter

 

1- Go to PROJECT MANAGEMENT and create a new PROJECT an a new FIT SUBPROJECT.

  • Add the dataseries created previously to the subproject.
  • Select the model created previously.
  • Keep all the default values in FitSetting.

2- Press Run button.

This Steps are described in the following Video Tutorial:

You can follow this tutorial in AFFINImeter, the Experimental Data and Binding model are stored in your own AFFINImeter account. If you hasn’t registered yet go to the AFFINImeter Software WebPage to get your account.

 

 

Ref: W. B. Turnbull, Divided we fall? Studying low-affinity fragments of ligands by ITC. GE Healthcare Life Sciences protocol, 2011, pp 1-11.

 

THANK YOU TO ALL PARTICIPANTS OF THE WIDEN-ITC OPEN DAY

THANK YOU TO ALL PARTICIPANTS OF THE WIDEN-ITC OPEN DAY: Over 100 researchers joined us to know more of the potential and latest advances in Isothermal Titration Calorimetry.

Speakers and invited researchers.
Speakers and invited researchers to WIDEN-ITC Open Day.

The WIDEN-ITC Open Day, has confirmed the awareness of the Scientific Community in exploring the full potential of the Isothermal Titration Calorimetry technique. Researchers from 18 countries attended on-site in Santiago de Compostela or via streaming this Open Day, consisting in 8 scientific talks and a tutorial of the ITC analysis software AFFINImeter (developed by the organizers of the workshop). Outstanding Pharmaceutical Companies (Lilly, Astra Zeneca,…..), microcalorimeters manufacturers (Malvern Microcal) and numerous Universities and Research Institutes worldwide participated in this interactive day,  dedicated  to share knowledge and exchange ideas around the present and future of ITC. The WIDEN-ITC Open Day discussed topics of actual interest like the current state of both ITC instruments and data analysis, common errors and misuses, and new analytical and technical developments to exploit the full potential of the technique and expand the application range of ITC. The morning session consisted in 6 invited talks of distinguished experts in the field: Adrián Velázquez-Campoy (University of Zaragoza, Spain), Phillipe Dumas (CNRS, Strassbourg, France), Eric Ennifar (CNRS, Strassbourg, France), Natalia Markova (Malvern Microcal), Niek Burma (Cardiff University, UK) and Daniel Ondo (Institute of Chemical Technology, Prague). The evening session of the Open Day was conducted by members of the scientific team of AFFINImeter, who presented 2 talks and an interactive tutorial to show the full potential of this new analysis software. Moreover, the workshop counted with the key participation of the invited researchers Margarita Bastos (University of Porto, Portugal), Maria Joao Moreno (University of Coimbra, Portugal), Luis García (University of Santiago de Compostela, Spain), Enrique García Hernández (National Autonomous University of Mexico) and Luis Pablo Calle (Analytical Technologies, Lilly, Spain)

Invited Speakers to WIDEN-ITC Open Day
Invited Speakers to WIDEN-ITC Open Day

We would like to specially thank all the presenters and invited researchers, who did an outstanding job sharing their expertise with us and again, to all participants for attending the WIDEN-ITC Open Day.

We would also like to extend our thanks to all the AFFINImeter members, to the University of Santiago de Compostela and the Galician Supercomputing Center (CESGA) for all the support during the organization of this event.

 

WIDEN-ITC: WIDENING KNOWLEDGE

WIDEN-ITC 2014

Worldwide reference research scientists on molecular interactions analyse technical advances in Isothermal Titration Calorimetry (ITC) at WIDEN-ITC.

Organised by AFFINImeter, a spin-off of the Universidad de Santiago de Compostela (USC), WIDEN-ITC will take place  September 10th at the Faculty of Physics of USC, with participation of about ten worldwide experts on specialised scientific software.

Advances in research for the development and analysis of chemical products, with applications in areas such as the pharmaceutical industry or material sciences, have been showing the shortcomings of available software for the efficient analysis of data from experimental techniques such as Isothermal Titration Calorimetry (ITC). The AFFINImeter software, a result of progress recently made to overcome these limitations, was launched in the international marketplace with a Software as a Service model, and is now being tested by 40 researchers from 25 countries.

The creators of AFFINImeter have invited at WIDEN-ITC some of the best worldwide reference scientists in ITC, with expertise in both current use and in the development of technological innovations that will shape the future of research with this particular technique. Prestigious researchers such as Philippe Dumas from University of Strasbourg, Adrián Velázquez from University of Zaragoza, and Ángel Piñeiro from USC and creator of AFFINImeter, among others, will work for three days with experts from industry, such as Natalia Markova from Malvern Microcal, to exchange knowledge and experience on the limitations of current software and new possibilities offered by technological advances.

Logo of the Workshop WIDEN-ITC 2014
Invitation to WIDEN-ITC, International Workshop on Isothermal Titration Calorimetry (ITC)

WIDEN-ITC will have an “Open Day” on the 10th of September at the Faculty of Physics of USC, which can be attended in person or online upon registration free of charge. The workshop will include a tutorial on the use of AFFINImeter, given by Eva Muñoz and Juan Sabín, researchers at AFFINImeter.

All the information about WIDEN-ITC is available on the website www.affinimeter.com.

WIDEN-ITC: Ensanchando el Conocimiento

WIDEN-ITC 2014

En WIDEN-ITC científicos de referencia mundial en la investigación de interacciones moleculares se reunen para analizar los avances de la técnica de Calorimetría de Titulación Isotérmica (Isothermal Titration Calorimetry, ITC).

Organizado por AFFINImeter, spin-off de la Universidad de Santiago de Compostela (USC), una decena de expertos mundiales en software científico participarán en el WIDEN-ITC, un workshop que se celebrará el 10 de septiembre en la Facultad de Físicas de la USC.
Los avances en la investigación para el descubrimiento y análisis de productos químicos, con aplicaciones en áreas como la industria farmacéutica o ciencia de materiales, han puesto en evidencia en los últimos años las carencias del software disponible para el análisis con técnicas experimentales como la Calorimetría de Titulación Isotérmica (ITC). Fruto de los avances para superar esas limitaciones es el software AFFINImeter, que acaba de presentarse en el mercado internacional con un modelo de Software as a Service, y que está siendo probado en su versión inicial por 40 investigadores de 25 países.
Los creadores de AFFINImeter convocan en el WIDEN-ITC a algunos de los expertos de referencia mundial en esta técnica, tanto en su uso actual como en el desarrollo de innovaciones tecnológicas que marcarán el futuro de la investigación con ITC. Prestigiosos investigadores como Philippe Dumas, de la Universidad de Estrasburgo, Adrián Velázquez, de la Universidad de Zaragoza o Ángel Piñeiro, creador de AFFINImeter e investigador de la USC, entre muchos otros, trabajarán durante tres días con expertos de la industria, como Natalia Markova, de Malvern Microcal, para intercambiar conocimientos y experiencias sobre las limitaciones del software actual y las nuevas posibilidades que permiten los avances tecnológicos.

Logo of the Workshop WIDEN-ITC 2014
Invitación a WIDEN-ITC, Workshop de Calorimetría de Titulación Isotérmica (ITC)

El WIDEN-ITC contará con un “Open Day” el 10 de septiembre, que podrá seguirse presencialmente en la Facultad de Físicas de la USC, o bien por internet desde cualquier lugar, con sólo realizar una inscripción gratuita. La jornada incluirá un tutorial para el uso de AFFINImeter impartido por Eva Muñoz y Juan Sabín, investigadores de AFFINImeter.
Toda la información del WIDEN-ITC está disponible en la web www.affinimeter.com.

Multiple Independent Sites: Advanced tools for a successful analysis.

Isothermal titration Calorimetry experiments of a ligand binding to a macromolecule with multiple independent sites

A successful Isothermal Titration Calorimetry (ITC) experiment requires the acquisition of high quality experimental data together with a careful analysis. Choosing the right binding model to fit the ITC isotherm is critical in order to get the true thermodynamic profile of the interaction. Often, the main limitation to achieve good results arises when the evaluation software lacks of the mathematical model that best describes our binding experiment. A good example is the case of a ligand binding to a macromolecule with multiple independent sites, i.e ligand – DNA interactions (1). Until now the readily available mathematical models to fit such experiments was limited to one or two sets of “n” independent identical sites; frequently, these models offer a poor description of the interaction due to the inherent higher complexity of the system, where many distinct binding equilibria coexist.

 

Multiple Sets of Independent Sites

AFFINImeter ITC offers an unlimited number of user-defined binding models. Particularly, it counts with a feature to easily design models based on multiple independent binding sites. Here, a model with a number of sets of independent sites can be created with no limitation in the number of sets or sites. Noteworthy, the number of sites in each set can be considered as a fitting parameter throughout the data analysis. As an illustration, the following figure shows the reaction parameters of a model generated with AFFINImeter that describes a ligand binding to a receptor having 3 sets of sites, each set having an unknown number of sites. Fitting the experimental data to such model yields the microscopic association constant (K) and the change in enthalpy (ΔH) of the ligand binding to each site type, and the number of sites in each set (n).

Scheme of the interaction of a ligand with a multisite receptor
Reaction parameters table of a model that describes the interaction of a ligand (in syringe) with a multi-site receptor (in cell) having 3 sets of independent sites. Note that the option “Fit” was checked to consider the number of sites (n) as fitting parameters.

These binding models, described by numerous variable parameters, may end up in an over-parameterized fitting function. Thus, the best strategy to achieve a robust and consistent analysis involves the global fitting of several ITC curves acquired under different experimental conditions. In this sense, AFFINImeter also supports global fitting of multiple isotherms wherein parameter linkage between curves is used to decrease the relative number of estimated parameters per experiment.

References:
(1) Methods 2007, 42, 162–172.

Use of precise standard reactions for Isothermal Titration Calorimeter validation

Use of  precise Standard Reactions for Isothermal Titration  Calorimeter Validation

Many published papers report inconsistent thermodynamic values of the same interactions between chemical reactants or macromolecular binding. One of the reasons for this discrepancies is the difficulty of repeating the same conditions in the ITC experiments (buffer, pH, concentrations, ionic strength, source of the materials…). But users start to be more aware that some systematic errors of the calorimeters may also have an important effect in the reported values.

For instance, the interaction between 4-carboxybenzenesulfonamide and bovine carbonic anhydrase II is considered a standard reaction to be measured by ITC and its enthalpy has been measured by 14 operator using different calorimeters (1). The resulting value considering all these independent measurements is -10.4±2.5 kcal·mol-1. The error of the enthalpy is surprisingly high and significantly higher than those typically reported for ITC measurements.

Baranauskiené and co-workers (1) suggest the use of precise standard reactions for  Isothermal Titration calorimeter validation after the calibration. The table below shows the series of chemical reaction they propose as standards where the enthalpy of binding has been determined to high precision and the reagents are readily available from commercial sources.

 

table-chemical-reactions-calorimeters-reference

They also used these standards reactions to compare the results obtained with different micro calorimeter. Their study concluded that Microcal calorimeters are more reliable than TA Calorimeters; and the most recent Microcal ITC200 is less accurate than Microcal VP-ITC. Nano ITC-III calorimeter results were very reproducible, but enthalpy values were systematically underestimated. To learn more about Isothermal Titration Calorimeter validation,, visit the references from where this article was taken.

VP-ITC-NanoITC-ITC200-calorimeter-comparison-entalphie
Enthalpy of the interaction between Tris base and HNO3 as a function of temperature and measured in different ITC calorimeter. Literature values were taken from (2).

References:

(1) Int. J. Mol. Sci 2009, 10, 2752-2762.

(2) Handbook of proton Ionization Heats Wiley-Interscience: Hoboken, NJ, USA, 1979

How to design an Isothermal Titration Calorimetry experiment?

Isothermal Titration Calorimetry Experiment Simulation

 

 theoretical-isotherm-Microcal-VP-ITCspecies-concentration-isothermal-titration-calorimetry-experiment

The Simulator tool available in AFFINImeter is completely free under registration. This is currently the only alternative to design complex Isothermal Titration Calorimetry (ITC) experiments. The Simulator allows plotting ITC curves (evolved heat as a function of the system concentration) together with a phase diagram of the different chemical species that are present in the solution regardless the complexity of the interaction mechanism between the involved molecules.

Scheme of a ITC Experiment
Isothermal Titration Calorimetry Experiment

 

Avoid Trial and Error Assays

Using the AFFINImeter Simulator you will be able to pre-visualize the results of an experiment, provided that you have an approach for the interaction mechanism of your molecules and of the corresponding thermodynamical parameters. This tool will guide you in the optimization of the most advantageous combination of experimental parameters: the concentration and location (in the sample cell or in the syringe) of your compounds, the injection volume and the number of titrations; thus avoiding trial-and-error assays and saving time, reactants and money.

This tool is also useful to set the conditions under which the distribution of chemical species meet some special requirement (for instance, the solution dominated by a given chemical species). It can also be used for didactic purposes since it helps to illustrate how a chemical species can be displaced by another, to explain the difference between cooperative and non-cooperative processes or to explain the effect of endothermic and exothermic processes.

 

Applications in Drug Discovery

Isothermal Titration Calorimetry is a key technique in the development of drugs since it assess the affinity between molecules. The most typical application is to determine the free energy of interaction between proteins and inhibitors. The AFFINImeter simulator tool allows simulating the displacement of a weak ligand by a strong ligand as a function of the concentration of the compounds involved in the experiment.

Advantages of the Simulator

Introduce your personalized thermodynamic model directly in chemical language (reaction scheme) and an estimation for the corresponding thermodynamic parameters. Even the most advanced models are easy to implement. Through the model builder AFFINImeter offers an unlimited amount of thermodynamic models for Isothermal Titration Calorimetry data analysis. If the model required for your system is not available, please, do not hesitate to contact us and we will try to implement it.

Sequential Binding Sites Model
This is a Sequential Binding Model that considers the free species of both solutes M and A, plus the hybrid complexes with stoichiometries (1:1, 1:2, 2:1, 2:2).

 

Start Using the Simulator

The AFFINImeter Simulator is free under registration. To learn how to use it, please read this tutorial.

The Model Builder is a versatile tool to translate binding interactions into mathematical models

The Model Builder is one of the novel features of AFFINImeter.  Through the model builder AFFINImeter offers an unlimited amount of thermodynamic models for ITC data analysis. The overall binding equilibria within the species involved in the experiments is easily drawn by the user directly in chemical language. Then AFFINImeter translates the resulting reaction scheme into robust  binding  models to be used to isotherm ITC simulation or to perform Isothermal Titration Calorimetry curve fitting.

The model builder is a versatile tool, it allows to design models involving up to three different species (i.e. the case of two ligands that compete with each other to bind a macromolecule) and has the advantage to selectively place them in the  syringe cell and/or in the calorimetric cell. 

Binding Reaction Scheme of a competitive interaction
Reaction Scheme of a Competitive Binding Interaction

It also allows the design of models for dissociation, ranging from simple homodimers to higher-order oligomers. During the model construction no mathematical equations are required, once the whole set of binding interactions is defined by the user in the reaction builder, AFFINImeter internally generates the system of equations that define the reaction scheme proposed. The new model (reaction scheme and equations) is saved  internally by AFFINImeter and listed in the user’s database so that can be utilized anytime so simulate or fit data.

AFFINImeter Reaction Builder
Competitive binding model builded with the AFFINImeter tool

AFFINImeter-ITC offers an exclusive unlimited amount of personalized model families including

  • Unrestricted Competitive Sequential Binding with no limitation in the stoichiometry of the binding model.
  • Competitive Multiple and Independent Sets of Identical and Independent Sites.
  • Dissociation of any chemical species including homogeneous n-mers, heterogeneous complexes and even micelles.

With this extensive offer of model families the user will be able to perform the thermodynamic characterization of a vast variety of biological and physicochemical processes from ITC measurements. A few examples of classical and new applications of ITC experiments that you can analyze with AFFINImeter are:

  • Protein-ligand or host-guest complex formation with unlimited stoichiometries
  • Competition of different molecules to occupy a given binding site even for high order complexes
  • Binding between ligands and polymers or large macromolecules with any number of (independent) sets and/or sites
  • Dissociation/aggregation of supramolecular heterogeneous species including protein oligomers
  • Structural and thermodynamic information of large aggregates, including micelles: aggregation number, enthalpy of formation, Gibbs energy and dilution heat of monomers and aggregates