When investigating molecular interactions, equilibrium dissociation constants (KD) are commonly used as a measure of the affinity of two molecules for each other. In other words, the KD is a direct measure of the strength of an interaction. Determining the KD is very important to evaluate the biological relevance of an interaction, such as in the study of fusion proteins, DNA-protein binding and for standard protein analysis.
However, obtaining only a KD value does not provide independent information on the kinetics of the molecular interaction. In other words, KD does not tell you how quickly the two molecules bind (i.e., the association rate constant or "on-rate" of the interaction), or how quickly the molecules dissociate (i.e., the dissociation rate constant or "off-rate" of the interaction). Indeed, molecular interactions that have the same KD can have widely different on- and off-rates (Fig. 2).
The kinetics of a molecular interaction are very important for drug discovery and development1, 2, 3, 4. The off-rate is especially important, because it tells you how long the compound binds to the ligand or target. A low off-rate would mean the drug could be formulated for less frequent dosing, whereas a high off-rate might require multiples doses per day. Given how important patient compliance is for treating many diseases—such as chronic diseases5 and acute infections—ensuring a compound can be used in a convenient dosage regimen is vital to drug development. Many studies have shown that patients are less likely to miss a dose when they are taking a drug once a day compared with twice a day, and a once-weekly dose results in even better compliance.
Kinetic data can also help reveal a compound's mechanism of action6, 7, 8 by determining whether the compound is interacting with, for example, one or multiple binding sites on the target9. This information is important for a number of reasons. For example, while most enzyme targets have a unique active site, they typically share other structural features with other enzymes. Interacting with such an allosteric site10 could lead to unwanted reactions with other enzymes. Alternatively, a researcher might be looking for an allosteric interaction to activate or modulate the target.
Surface plasmon resonance (SPR) can be used to determine both kinetic/thermodynamic and equilibrium data, as well as the concentration of active compounds, to:
No other biophysical technique provides such comprehensive information in real time in only one system.
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