Application of microcalorimetry in drug discovery at Exelixis
In this report, we illustrate the utility of calorimetry in making better laboratory decisions. Examples will include improved protein construct selection for scale-up; direct measurement of the effects of mutations and post-translational modifications on protein stability; rapid optimization of solvent formulation; direct measurement of substrate and inhibitor binding affinity; determination of the mode of inhibitor binding; characterization of protein-protein interactions; and improved structural biology efficiency, when used in conjunction of other biophysical methods.
Introduction
Understanding the molecular details of drug-target protein interactions is a critical component of the drug discovery process in the modern pharmaceutical industry. We have put in place a comprehensive set of highly integrated biochemical and biophysical methods to better characterize the target protein and its interactions with inhibitors. These techniques enable us to identify chemical instability (oxidation, deamination etc.), proteolytic or chemical degradation, post-translational modification, and physical instability such as surface denaturation, soluble aggregation, and precipitation. More complete routine characterization of the protein of interest informs the development of better formulations, as we are able to quantitate the effects of formulation components on protein stability. A critical component of our biophysical arsenal is the ability to investigating a protein’s thermodynamic properties by ITC and DSC. Both techniques are based in well established fundamental principles. ITC provides thermodynamic data used to confirm the binding model, as well as quantitation of binding enthalpy (ΔH), entropy (TΔS), free energy (ΔG) and binding association constant Ka. DSC measures the heat changes associated with thermal denaturation of the target protein and has been extensively used in understanding protein folding and unfolding. The degree of stabilization conferred by compound binding is often related to the affinity of the interaction. The thermodynamics associated with unfolding (change of Tm, ΔCp, and ΔH,) reveal information about the properties of compound binding.1,2 Classically, both measurements required a significant amount of protein and commitment of significant operator time, as the instrumentation required manual operation. These factors limited wide application of these measurements. Recently, MicroCal LLC. introduced two new instruments, the AutoITC and AutoDSC, with significantly improved sensitivity, greatly reduced sample consumption, robotic automation and user friendly software packages that greatly simplify experimental set-up and data analysis. In this paper, we will present examples of disciplines in small molecule drug discovery where this new instrumentation has allowed us to improve the efficiency of our processes.
