Category: Tutorial & Examples

Competitive Binding Assay

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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.

 

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

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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

How to perform a neat Isothermal Titration Calorimetry experiment?

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In a Isothermal Titration Calorimetry experiment, If the injected volume or any of the concentrations is too small, or if the ratio between both concentration values is not appropriate, then the signal-to-noise ratio will be low and the uncertainty of any result will be high.

Scheme of a ITC Experiment
Isothermal Titration Calorimetry Experiment

Determinant factors of poor quality results in Isothermal Titration Calorimetry Experiments

The control of these factors might be limited by the available amount or the corresponding solutes (they can be expensive or difficult to synthesize/purify). If the total number of titrations is low then the solute in the sample cell will not be saturated and the quality of the results will be poor. Additionally, in reactions for which multiple chemical species can be formed it is always better to simultaneously fit several experimental data series, each focusing the sampling in a different concentration region which is more sensitive to any of the species.

Advantages of prior Isotherm Simulation

The simulator tool provided by AFFINImeter allows you to test the effect of the parameters listed above on your experiment in order to optimize its design, thus saving time and samples.

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