Addressing the needs of drug discovery with Microcal PEAQ-ITC

Dr Michael Caves, India Business Development Manager, Malvern Instruments, in this article describes one of many key features of new Microcal PEAQ-ITC used to improve the efficiency of drug discovery and development

Measurements and characterisation of binding interactions between proteins and low-molecular weight ligands are fundamental for drug discovery. Among the most recognised challenges in characterising binding interactions are
(1) The need to accurately assess a wide span of binding affinities (KD) and
(2) Accurately rank and characterise low-molecular weight (LMW) ligands based on affinity, mechanism of action, and energetics of interaction.

Isothermal titration calorimetry (ITC) directly measures heat released or absorbed in a binding event, providing means for studying protein-small molecule interactions in solution without the need for labelling or immobilisation. A highly sensitive ITC instrument and properly designed experimental conditions make it possible to account for inaccurately assessed protein concentration or inactive protein fraction, and to account for imprecise concentration of compound solutions. From quality control (QC) to assay development and lead optimisation, ITC has a role in improving our understanding of biochemical data. Normalising for protein or compound concentrations and accounting for inactive populations of protein can improve decision-making processes, one example being a more careful assessment of changes in enthalpic contributions to binding, which are critical in many best-in-class drug studies.

Simultaneous affinity determination for isomers and enantiomers

In late-stage drug discovery, a significant number of compounds are synthesised as mixtures of enantiomers or isomers. In many of these cases, the compound mixture can be separated by high-performance liquid chromatography (HPLC) and one or more isomers can be tested individually. However, in many cases, particularly those involving mixtures of enantiomers, separation can be difficult and time-consuming, and in these cases the mixtures must be tested directly. Biochemical assays primarily provide information on the tightest binding component in the mixture. In contrast, ITC can provide binding information about both the strong and the weaker binder simultaneously in a single experiment.

The biphasic binding isotherm in Figure 1 represents an example how the ITC data may look for the injection of an enantiomeric ligand mixture into a target protein in the cell. Similarly, biphasic isotherms are observed when high affinity ligands are intentionally mixed with weaker ligands as a form of competition experiment to determine KDs outside the range of direct measurements. This differs from the more typical approach of injecting the tight binding ligand into a mixture of the target and the weak inhibitor but has the advantage that both inhibitors can be resolved simultaneously and without prior knowledge of the interaction parameters of the weak interaction.

Figure 2: Raw and normalised heat plots for the titrations of the 1:1 mixture of EZA and FUR into bCAII. The titrations were carried out at 160 µM total concentration of the compounds in the syringe and 10 µM concentation of bCAII in the cell (left) or at 100 µM concentration of the compound mixture and 5 µM concentation of bCAII in the cell (right). 18 × 2 µl injection were made and the buffer was PBS, 2 v/v % DMSO at 25°C

To demonstrate the utility of the MicroCal PEAQ-ITC Analysis Software for the analysis of complex binding isotherms we have performed experiments with mixed ligands (ethoxzolamide (EZA), and furosemide (FUR)) in the syringe injected into a target protein (Bovine carbonic anhydrase II, bCAII) in the cell. The data are shown in Figure 2 and represent the type of biphasic isotherm data that is expected for an enantiomeric mixture or a ‘syringe’ competition experiment.

These experiments demonstrate the level of resolution that can be achieved using the MicroCal PEAQ-ITC and the potential for quantitative characterisation of the binding of mixtures. The fitting of complex ITC data, and in particular the fitting of these competitive experiments, has been made simpler in the MicroCal PEAQ-ITC software than in analysis software previously available on earlier models. While the input of good initial guesses into the fitting software is always a good start, the current software contains more appropriate numerical boundaries increasing the chances of a successful fit and minimising the risk of the fitting process getting trapped in a local minima. Fitting these data also benefited from combining the Simplex fitting and Marquardt-Levenberg fitting approaches available in the software. More specifically, using the Simplex approach at the start and finalising using the Marquardt-Levenberg fitting algorithm. The thermodynamic parameters obtained using this approach with the ‘two-sites’ model and ‘ligand in cell’ setting are summarised in Table 1.

Summary and conclusions

The new MicroCal PEAQ-ITC instrument along with improved signal stability, mixing and signal-to-noise characteristics has data analysis software well-suited for use in the biophysical laboratories involved in small-molecule drug discovery.

The analysis is completely automated, minimising user subjectivity in assessing data quality and the analysis process. Data quality assessment and fitting is performed rapidly allowing for analysis of large data sets of 50 or more experiments in seconds.

Key additional features of the new MicroCal PEAQ-ITC Analysis Software package allow for the determination of the active concentration of the target protein or the ligand concentration or both when the appropriate controls are used. This provides for the more accurate determination of the affinity and thermodynamic allowing for rigorous structure-activity relationship in hit validation and lead optimisation programmes.

In addition, the new ITC data analysis software has tools that simplify the fitting of complex binding isotherms that may be observed when titrating with enantiomeric mixtures, some competition experiments or with targets with more than one binding sites.