Kinetic Characterization

IC50 is widely-used as a measurement of potency in drug discovery but has limitations in predicting in vivo drug interaction. IC50 is dependent on the experimental conditions under which the value is determined, which makes the IC50 value only meaningful at the substrate concentration and incubation period under which it was measured. In contrast, the equilibrium dissociation constant (KD), a measure of affinity, is an intrinsic measure of the strength of binding between a target and analyte that does not change regardless of substrate concentration or run time. Kinetic characterization enables flexibility and time savings in comparing different systems that IC50 values cannot provide.

In addition to measuring KD, drugs with slow dissociation rates (Koff) often have extended duration of action, and affinity may not be an accurate proxy to discover which compounds will provide beneficial efficacy durations. While slow dissociation is often connected with high binding affinity, this is not always the case, and without a dependable kinetic binding platform, it’s impossible to know for sure. By characterizing on and off rates at the same time as affinity, compounds with desirable kinetic profiles for target binding can be chosen.

Many compounds can bind to numerous off-target proteins as well as the intended target. Monitoring the dissociation rates of the compound from all possible targets may be advantageous. Kinetic profiles can differentiate compounds with improved toxicity and selectivity, by enabling selection of those that have both slow off-rates for chosen targets and fast off-rates from unintended ones.

Agile R100 features:

  • Sensitive kinetic binding measurements kon, koff, and KD
  • Characterization of small molecule fragments ≥10 Da
  • Functionalization with just 0.5 ng of material
  • 11-log dynamic range from fM to mM provides a large working range of concentrations on a single platform
  • Easy gathering of affinity data enables fast turnarounds for structure-activity relationship (SAR) research

 

label-free Agile R100 kinetic characterization

In the figure above, an overlay plot displays a full kinetic characterization using COOH Agile biosensors. A concentration series of small molecule SPD304 is measured against immobilized TNFα, a cell signaling protein used as a therapeutic target. These curves show the concentration dependence of the biomolecular interaction, and the evaluation software provides kon, koff, and KD.

Additional Literature: