Ki, IC50 and EC50: What Binding and Potency Figures Actually Measure

Almost every claim about a research peptide’s selectivity rests on one of three numbers. They appear side by side in papers, in review tables and in supplier copy, and they are routinely treated as interchangeable measures of how strong something is. They are not. They measure different quantities, under different experimental conditions, and only one of them is a property of the molecule and its target alone.

This is the reading-the-literature companion to our note on peptide nomenclature: what the numbers next to the names mean.

Kd and Ki: affinity

The dissociation constant, Kd, describes how tightly a molecule and its binding site hold together at equilibrium. It has units of concentration, and the value is the concentration at which half the binding sites are occupied. A lower number means tighter binding: a Kd of 1 nM is a hundredfold tighter than 100 nM.

Most published affinity data for peptides comes from competition binding rather than direct measurement. A radiolabelled or fluorescent reference ligand is bound to the receptor, the test compound is added across a concentration range, and the concentration that displaces half the reference ligand is recorded. That raw value is an IC50 — it depends on how much reference ligand was used and on the reference ligand’s own affinity.

Ki is that IC50 corrected for those two variables, using the Cheng–Prusoff relationship. The correction is what makes Ki comparable between laboratories: two Ki values for the same receptor are meaningfully comparable, whereas two competition IC50 values from different assay setups are not.

The practical rule when reading a table: if the column is headed Ki, the numbers can be compared across sources. If it is headed IC50 in a binding context, they can only be compared within the same assay.

EC50: functional potency

EC50 is a different kind of measurement. It is the concentration producing half of the maximal response in a functional assay — second messenger accumulation, reporter output, calcium flux, receptor internalisation, whatever the assay reads out.

Because it reports a response rather than occupancy, EC50 folds in everything between binding and readout: receptor expression level in that cell line, coupling efficiency, amplification through the signalling cascade, assay incubation time. A receptor expressed at high density in a recombinant line can produce a full response with a small fraction of sites occupied, which shifts EC50 well below Kd. The same compound in a low-expression system can show an EC50 above its Kd.

So EC50 is a property of the compound in that assay, not of the compound alone. An EC50 without the cell system and readout stated is a number without a referent.

Why the distinction changes conclusions

Selectivity claims are the place this matters most. A statement that a peptide is “selective for one receptor subtype” is usually derived from a ratio of two numbers, and the ratio is only meaningful if both numbers came from the same kind of measurement.

A compound can bind two subtypes with similar affinity and activate them with very different potency, because the two subtypes couple to different pathways with different amplification. Reported the other way round, a compound can look selective on affinity and behave non-selectively in a functional assay. Both patterns appear in the melanocortin literature, which is why subtype selectivity in that family has to be read carefully rather than taken from a headline figure.

The same caution applies to the incretin receptor agonists, where a molecule’s relative activity at two or three receptors is the entire point of its design and the reported ratios shift depending on which assay produced them. See incretin receptor pharmacology.

Efficacy is a separate axis from potency

Potency is where the concentration–response curve sits on the concentration axis. Efficacy is how high it rises. A compound can be highly potent and only partially efficacious: it occupies the receptor at low concentration but never produces a full response no matter how much is added.

Two compounds with identical EC50 values can therefore behave very differently, and a table listing only EC50 does not distinguish them. The maximal response, usually reported as a percentage of a reference agonist’s, is the missing column. A partial agonist and a full agonist are different pharmacological tools even when their potencies match.

Biased agonism, briefly

A receptor can signal through more than one downstream pathway, and a ligand can favour one over another. When that happens, a single EC50 is not merely incomplete — it is pathway-specific. Two papers reporting different EC50 values for the same peptide at the same receptor may both be correct and simply be reading different outputs.

This is one reason apparently contradictory literature is often not contradictory. It is also a reason to record which readout a number came from when you write it down.

Reading a number properly

When a figure appears without context, the questions that make it usable are:

• Is this affinity (Kd, Ki) or function (EC50)? A binding IC50 and a functional EC50 are not the same measurement.
• What system: recombinant cell line, native tissue, which species’ receptor? Sequence differences between species change affinity.
• What readout, and over what incubation?
• Is the maximal response reported alongside the potency?
• Is the comparison being drawn from numbers of the same type, measured the same way?

Where a supplier or a review states a selectivity ratio without answering those, the ratio is a claim rather than a measurement. Where the underlying paper answers them, the number is usable — and usually more interesting than the summary made of it.

Two further notes bear on how these figures behave for peptides specifically: half-life and analog modification, since a compound’s persistence in an assay affects what a fixed-incubation EC50 reports, and cyclisation, which changes affinity by constraining conformation.

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