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Through binding a drug to a target, the effect of that target is changed, and therefore a patient’s disease state is changed. Based on this logic, one of the early steps for a drug discovery program is to confirm that an active molecule physically binds, or engages, the intended target. Drug binding can be described as an equilibrium. The drug (D) binds the target (T) to form a drug-target complex. Most drugs bind a target reversibly, so drug binding is an equilibrium process. As an equilibrium, we can quantify the equilibrium through either the association equilibrium constant (Ka for the forward process) or the dissociation equilibrium constant (Kd, the reverse process). Almost always, researchers focus on Kd. Two different common binding assays can determine Kd values for a drug-target pair.
One binding assay method is surface plasmon resonance, or SPR. In SPR, a target protein is bound to a reflective gold surface. Light reflection measurements are made. The target is then exposed to the ligand, the drug, to allow complex formation. Light reflection properties of the complex are compared to the unbound target to determine the effect of binding. SPR is used to determine Kd as well as the rate constants kon and koff, so SPR also reveals the kinetics of binding, the rate of binding.
The second method is isothermal titration calorimetry, or ITC. In ITC, the target and ligand are mixed and temperature changes arising from the release of binding energy are measured. These temperature changes allow determination of Kd as well as the thermodynamic changes – enthalpy, entropy, and free energy. The stoichiometry of binding – that’s the number of ligand molecules binding to the target – can also be determined.
SPR & ITC are both examples of in vitro assays. Older binding assay technologies require the drug or analyte compound to have a radioactive label. SPR & ITC do not and are therefore “label-free” binding assays. SPR & ITC do require access to the purified drug target or at least the binding domain portion of the target, which may be an option for some receptors. There are cellular assays that measure binding, and these assay formats are becoming more broadly available. Overall, SPR is often the method of choice for rapidly testing target engagement for a large collection of molecules.