Pursuing a reliable calculation of binding affinity.

The relevance of uncertainties.

 

The reliable determination of binding constants (Ka) and enthalpies (ΔH) from Isothermal Titration Calorimetry depends, among other aspects, on the quality of the thermogram recorded where a high signal-to-noise ratio is pursued to get a trusty isotherm. Low signal-to-noise ratio conducts to less precise integration of the thermogram peaks and, consequently, to a binding isotherm defined by points with larger related uncertainties. Logically, the uncertainty associated to the integral points will significantly affect the determination of the thermodynamic parameters and should not be set aside through the data analysis.

Here´s an illustrative example of two ITC experiments of the interaction between Carbonic Anhydrase II (CA) and 4-carboxybenzenesulfonamide (4-CBS) that yielded thermograms of different quality (Fig 1*).

Fig.1 ITC titrations of 4-CBS into CA. a) Exp1, higher signal-to-noise ratio; b) Exp2, lower signal-to-noise ratio.

Data fitting to a 1:1 binding model was performed for both experiments and for comparison purposes the analyses were conducted twice, taking into account the uncertainties associated to the calculation of the peaks integral (weighted fitting) and not considering them (all the points having the same weight).
The results show a larger discrepancy in the Ka values obtained from Exp2, depending on whether or not the uncertainties are included in the fitting. Besides, the Ka obtained from the weighted fitting of Exp2 is significantly closer to the corresponding Ka value of the weighted fitting of Exp1 (Figure 2).
Fig.2 KA, DH and rM (correction parameter for CA concentration in the cell) values from fitting of Exp1 and Exp2 a) without uncertainties and b) with uncertainties. The bar diagrams show the comparison between KA values of Exp1 and Exp2.

 

The main origin of this difference is the large uncertainty associated to the mid-titration point, which consideration has an effect on the final Ka value (Figure 3).
Fig.3 Fitted isotherm from Exp 2. Redline: theoretical curve calculated when considering uncertainties; black line: theoretical curve calculated with no uncertainties.

 

Being aware of the relevance of weighting data points based on their uncertainty AFFINImeter includes, into the raw data processing step, the automatic determination of uncertainties in the peaks integration and the possibility to perform weighted fitting for a more reliable analysis. Another good reason to work with AFFINImeter!

 

Solving complex interactions with AFFINInimeter: Competing ligands binding to a multiple site receptor

Isothermal Titration Calorimetry (ITC) is a versatile technique with the potential of deconvoluting the various binding events that may coexist in complex interactions. In this sense, a major drawback has been the lack of mathematical models and computational tools to properly analyze such experiments. AFFINImeter counts with an advanced functionality, the “Model Builder” with which researchers can easily design their own binding models through the combination of distinct (coupled) binding equilibria, to obtain thermodynamic and structural information from complex ITC experiments.

Two Competing ligands binding to a receptor with two sites

As a demonstration of the potential of AFFINImeter and its “Model Builder”, the analysis of an ITC experiment involving a receptor “M” (biomacromolecule) with two different binding sites (s1 and s2) that may accommodate two competing ligands (“A” and ”B”) is presented here.

Download complete case here: Competitive Binding case of use

Expanding the range of applications of ITC in the Pharmaceutical Industry with AFFINImeter: A practical Case.

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.
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

  1. 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.

  2. For a detailed description of the protocol contact us at support@affinimeter.com

Global fitting analysis of a protein-ligand binding experiment

Global fitting analysis of a protein-ligand binding experiment

A few weeks ago AFFINImeter launched an Isothermal Titration data Analysis challenge of the analysis of a protein-ligand binding experiments. The participant had to globally analyze a set of Isothermal Titration Calorimetry experiments using AFFINImeter and get the thermodynamic and structural parameters of the interaction between both molecules (the receptor protein and the ligand).

The participants in this contest had the opportunity to demonstrate their ability to propose the right model for a given binding isotherm as well as to get the corresponding parameters upon fitting using AFFINImeter. On their side, less experienced participants had the opportunity to:

The results recently published by Henlz et al in [Methods 59 (2013) 336-348] were taken as a reference to generate the set of ITC isotherms selected for this contest.

(more…)

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.

 

 

 

Global Fitting Analysis in ITC Displacement Titrations with AFFINImeter

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. AFFINImeter counts with advanced tools, like the global fitting of multiple dataseries and the analysis of isotherms registered under unusual experimental design, which can facilitate the analysis and expand the range of applications of isothermal titration calorimetry experiments. As an illustration, herein we present a displacement titration assay to determine the thermodynamics of HIV-protease with indinavir, a high affinity binder, and with acetyl-pepstatin, a weaker ligand. Using AFFINImeter a global analysis of four isotherms was performed describing: HIV-protease binding to indinavir (I) or to acetyl-pepstatin (II): HIV-protease binding to indinavir incorporating acetyl-pepstatin in the cell (III) or in the syringe (IV).

Isothermal Titration Calorimetry is one of the most commonly used approaches to obtain affinity and thermodynamic data of molecular interactions and has become a routine method in the pharmaceutical industry.1 Isothermal titration Calorimetry is applicable to numerous interacting systems, as long as a detectable heat change is produced during complexation, covering an important range of binding affinities (106 ≤ KA ≤108 M-1). Nevertheless, standard ITC experiments present some limitations in the case of very low- or very high-affinity interactions (i.e. affinities in the low millimolar or high nanomolar range, respectively). High affinity interactions (KA ≥ 109 M-1) yield square-shaped isotherms whose fitting yield accurate values of the binding enthalpy but only estimates of the association constant. Attempts to recover a sigmoidal shape requires the use of very low concentrations of the interactants that, in most cases, is not feasible in the practice (the minimum concentration that will typically cause a confidently measurable heat change for a 1:1 interaction is about 10 μM). On the opposite, low affinity interactions should be studied at high concentrations and this requirements is often a serious limiting step due to various potential reasons like limited solubility and/or availability of the sample molecule, or the existence of aggregation processes at the required concentration. In both high- and low affinity systems these experimental drawbacks can be circumvented by using the ITC displacement method.2,3 Here, the receptor is titrated with a high affinity ligand, but in the presence of a weaker ligand in the sample cell that competes for the complexation with the receptor (figure 1). With this experimental set up the apparent affinity of the strong ligand is “artificially” lowered, obtaining a sigmoidal isotherm that yields more accurate binding data. When the goal is to obtain the thermodynamic parameters of an ultra highaffinity system, a titration with the weaker binder is performed first to obtain the corresponding affinity constant and enthalpy (KA-weak and H-weak). These values are required for the analysis of the ITC displacement experiment, where a competitive binding model is used to estimate the thermodynamic parameters of the tight binding (KA-tight and H-tight). Analogously, when the goal is to obtain information of an ultra low- affinity system a direct ITC titration of the receptor alone with a ligand of higher affinity is performed. The resulting KA-tight and H-tight are then incorporated in the analysis of the isotherm from the ITC displacement assay.

This 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.
1 G. Holdgate, S. Geschwindner, A. Breeze, G. Davies, N. Colclough, D. Temesi, L. Ward, Biophysical Methods in Drug Discovery from Small Molecule to Pharmaceutical. Protein-Ligand Interactions. In Methods in Molecular Biology 2013, 1008, pp 327-355.
2 A. Vellazquez-Campoy and E. Freire, Isothermal titration calorimetry to determine association constants for highaffinity ligands. Nature protocols 2006, 1, pp 186-191.
3 W. B. Turnbull, Divided we fall? Studying low-affinity fragments of ligands by ITC. GE Healthcare Life Sciences protocol.

 

Download the case of use here

Isothermal Titration Calorimetry
Global Analysis in ITC Displacement Titrations with AFFINImeter