The analysis of isotherms is the more direct way to calculate binding constants for molecular interactions. Isotherms can be obtained using different techniques (ITC, SPR, NMR, Uv-vis, IR, Fluorescence, Circular Dichroism…) and at different experimental conditions.
1. The global fitting approach allows to simultaneously analyze several isotherms obtained by different biophysical techniques and/or at different experimental conditions in a very accurate manner.
2. Many interacting systems do not bind with a simple 1:1 model, more complex binding model can be designed to address complex interaction
3. Using tools to globally analyze isotherms obtained by different biophysical techniques is the most reliable method to characterize binding interactions by the orthogonal approach.
Click here to star using AFFINImeter and to start to create your own binding model:
Many Drug–receptor interactions are characterized by complex binding modes that are far away from the behavior of a standard 1:1 model. This is the case of Heparin (Hp), one of the most commonly prescribed anticoagulant drugs, which exerts its effect through its interaction with the serine protease Antithrombin (AT-III). Hp is a linear heterogeneous polysaccharide containing a specific pentasaccharide sequence that binds AT-III with high nanomolar affinity (responsible for the anticoagulant activity); but AT-III also binds other Hp sequences with lower affinity. Determining the content of AT-III binding pentasacchride in Low Molecular Weight (LMW) Heparins is a requirement for Pharmaceutical companies that manufacture this type of anticoagulants; due to the intrinsic heterogeneity of Hp, obtaining this information it is not straightforward (1).
We have developed a new protocol based on ITC and AFFINImeter to determine the content of AT-III binding pentasaccharide in Heparins, which is summarized in the following scheme:
New method based on AFFINImeter to determine the content of AT-III binding pentasacchride in LMW Hp: 1) use of a tailored binding model that describes the competitive binding between the pentasaccharide (A) and other low affinity sequences (B) with AT-III (M); 2) global fitting of several isotherms registered under different Hp and or AT-III concentrations where the parameters rA and rB (that account for the fraction of A and B in the Hp sample) are fitting parameters and common among the different isotherms.
This method illustrates the great potential of the model builder and global fitting AFFINImeter tools to develop protocols of practical utility in the Pharmaceutical industry (2). We have successfully validated the protocol in the analysis of unfractionated Hp and a series LMW Hp in collaboration with the Pharmaceutical company Laboratorios Rovi (http://www.rovi.es/).
References
Nandurkar H., Chong B, Salem H, Gallus A, Ferro V, McKinnon R. Low-molecular-weight heparin biosimilars: potential implications for clinical practice. Internal Medicine Journal, 2012, 44(5), pp 497–500.
Isothermal titration calorimetry (ITC) is an extremely sensitive technique to assess for the formation/disruption of complex chemical/biological species in solution. During the last years, the increase in instrument sensitivity as well as the reduction of the sample concentration required to perform experiments, have made possible to expand the application range of ITC, which is expected to continue growing.
Quality of the ITC Raw Data?
The amount and the quality of useful information that can be obtained from an ITC experiment depend on several factors including the purity of the samples, the concentration of the solutions prepared, the choice of injection volume and its length in time. The researcher handling the instrument is responsible for the appropiate selection of these variables as part of the experimental setup. They can be optimized on the basis of previous experience and also taking advantage of computational simulations. A key factor for this is that ITC is an incremental technique and so the results depend strongly on the injection volume employed to perform the experiment.
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”.
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”).
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.
