A graduate student building a comparison table for a family of receptor ligands pulls numbers from six papers. One compound is listed at 2 nM, another at 40 nM, and the table seems to show a twentyfold difference in strength. On closer reading, the first figure is a Ki from a competition binding assay, and the second is an EC50 from a reporter assay in an engineered cell line. The two numbers measure different things under different conditions. Put side by side, they produce a comparison that looks precise and means very little. Knowing what Kd, Ki, IC50 and EC50 each describe is the difference between a usable table and a misleading one.
Affinity: Kd and Ki
The dissociation constant, Kd, describes how tightly a ligand and its binding site hold together at equilibrium. It has units of concentration and equals the ligand concentration at which half the binding sites are occupied. A lower Kd means tighter binding: 1 nM is a hundredfold tighter than 100 nM.
Much published affinity data does not come from measuring Kd directly. It comes from competition binding. A labeled reference ligand, radioactive or fluorescent, is bound to the receptor, and increasing concentrations of the test compound are added. The concentration that displaces half the specific binding of the reference is recorded as an IC50.
That IC50 is not a property of the test compound alone. It depends on how much labeled ligand was used and on that ligand’s own affinity. The inhibition constant, Ki, corrects for both using the Cheng-Prusoff relationship, in which Ki equals the IC50 divided by one plus the ratio of labeled-ligand concentration to its Kd. After that correction, Ki values for the same receptor can be compared across studies. Uncorrected competition IC50 values from different assay setups cannot.
Function: EC50 and what it absorbs
An EC50 is a different kind of number. It is the concentration that produces half the maximal response in a functional assay, whether the readout is second-messenger accumulation, reporter gene output, calcium flux or receptor internalization. Because it measures response rather than occupancy, it absorbs everything between binding and readout:
- Receptor expression level in the cell system
- Efficiency of coupling to the signaling pathway
- Amplification along the signaling cascade
- Incubation time and assay conditions
In a cell line that expresses a receptor at high density, a full response may require only a small fraction of receptors to be occupied, which pushes the EC50 well below the Kd. In a low-expression system the same compound can show an EC50 above its Kd. An EC50 is therefore a property of a compound in a particular assay. Quoted without the cell system and readout, it has no fixed meaning.
A quick reference
| Figure | What it measures | Comparable across studies? |
|---|---|---|
| Kd | Equilibrium affinity, measured directly | Yes, for the same receptor and species |
| Ki | Affinity derived from competition, corrected for assay conditions | Yes, for the same receptor and species |
| IC50 (binding) | Concentration displacing half of a labeled ligand | Only within one assay setup |
| IC50 (functional) | Concentration inhibiting half of a measured response | Only within one assay system |
| EC50 | Concentration producing half the maximal response | Only within one assay system |
Potency is not efficacy
Potency describes where a concentration-response curve sits along the concentration axis. Efficacy describes how high it rises. A compound can be potent, producing its effect at low concentration in the assay, and still reach only part of the maximal response no matter how much is added. That is a partial agonist. Two compounds with identical EC50 values can therefore behave very differently, and a table listing only EC50 cannot tell them apart. The missing column is the maximal response, usually expressed as a percentage of a reference agonist.
One receptor, several pathways
Many receptors signal through more than one downstream pathway, and some ligands favor one pathway over another, a behavior called biased agonism. Where it occurs, an EC50 is specific to the pathway measured. Two papers that report different EC50 values for the same ligand at the same receptor may both be correct and simply be measuring different outputs. This is one reason apparently contradictory findings often are not, and a strong argument for recording the readout next to every number. Receptor-family comparisons that depend on these distinctions are discussed in GHRH and ghrelin receptor peptides compared.
Selectivity ratios need matched numbers
Claims that a ligand is selective for one receptor subtype are almost always ratios of two figures. A ratio is only meaningful when both figures are the same type, measured the same way. A compound can bind two subtypes with similar affinity yet activate them with very different potency, because the subtypes couple to pathways with different amplification. The reverse also happens.
A checklist for any number you copy into a table
- Is it affinity (Kd, Ki) or function (EC50, functional IC50)?
- What system produced it: recombinant cell line or native tissue, and which species’ receptor?
- What readout, and over what incubation time?
- Is the maximal response reported alongside the potency?
- Are all numbers being compared of the same type and from comparable methods?
- Was the concentration of the test material itself known accurately?
The last point is easy to overlook. Every one of these figures is a concentration, and a concentration is only as good as the amount of peptide actually present. A weighed solid includes counter-ions and water as well as peptide, so nominal concentrations can overstate the true value and shift apparent potency. The distinction is explained in peptide purity vs net peptide content, and related terms are defined in the research peptide glossary.
Questions
Is a lower number always better?
A lower Kd, Ki or EC50 indicates higher affinity or potency in that measurement. It says nothing about efficacy, selectivity or how the compound behaves in a different assay.
Can an IC50 be converted to a Ki?
For a competition binding assay, yes, if the labeled-ligand concentration and its Kd are known, using the Cheng-Prusoff relationship. Functional IC50 values are not converted this way.
Why do two labs report different EC50 values for the same compound?
Differences in receptor expression, cell background, readout, pathway and incubation time all move EC50. The values may both be valid for their own systems.
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