Receptor Binding Affinity Explained: Ki, IC50 and EC50

Open almost any peptide or small-molecule study and you will meet a wall of numbers: a compound reported with a Ki of 3 nM here, an IC50 of 120 nM there, an EC50 of 7 µM somewhere else. Understanding binding affinity Ki IC50 and EC50 values is one of the most useful literacy skills for anyone reading laboratory pharmacology, because these three parameters describe different things and are routinely confused. This article explains what each measures, how they relate, and how to read them critically in a research context.

What binding affinity Ki IC50 and EC50 values actually measure

At the heart of receptor pharmacology is a simple, reversible interaction: a ligand associates with and dissociates from a binding site. When that reaction reaches equilibrium, the balance between binding and unbinding is captured by the dissociation constant, Kd. A low Kd means the complex holds together well at low concentrations, which is what researchers mean by high “affinity.” Studies of hormone receptors, for example, have characterized binding as saturable and high-affinity, reporting apparent Kd values in the low-nanomolar range for a single binding population. Ki, IC50, and EC50 are all concentration values expressed in molar units (often nM or µM), and in every case a smaller number indicates that less compound is needed to produce the measured effect. That is the one intuition that carries across all three.

Ki: the inhibition constant

Ki is an equilibrium binding constant that describes how tightly an inhibitor or antagonist occupies a target, independent of the particular assay conditions used to measure it. Conceptually it is very close to Kd but applies to the inhibitor in a competitive setting. Because Ki is meant to be a property of the molecule-target pair rather than of the experiment, it is the value medicinal chemists prefer when comparing compounds across different laboratories. Modeling work has treated the target dissociation constant as a system-independent parameter and used it to reason about how affinity relates to selectivity across tissues and off-targets (Vlot et al., 2017). When a paper reports a Ki, it is making a claim about intrinsic affinity.

IC50: half-maximal inhibitory concentration

IC50 is the concentration of a compound that reduces a measured activity by half under the specific conditions of that assay. It is enormously common because it falls straight out of a dose-response experiment, but that convenience comes with a caveat.

Why IC50 depends on the assay

Unlike Ki, an IC50 is not a fixed property of a molecule. It shifts with the concentration of the competing substrate or agonist, the amount of target present, and other experimental details. This is why two studies can report different IC50 values for the same compound without either being wrong. Reviews of transporter assays have shown that IC50 estimates can vary substantially between laboratories and, in some systems, do not even follow the expected competitive relationship, underscoring that an IC50 is an operational readout rather than a pure affinity constant (Bentz & Ellens, 2021).

The Cheng-Prusoff bridge from IC50 to Ki

Because IC50 is condition-dependent and Ki is not, researchers often convert one to the other using the Cheng-Prusoff equation, first described in 1973. It corrects an observed IC50 for the substrate or agonist concentration used, yielding an estimate of the underlying Ki. Later analyses have refined this relationship, showing that the slope of the concentration-response curve matters and that the classic equation holds cleanly only when that slope is close to unity (Cheng, 2002). The practical takeaway for a reader is that an IC50 and a Ki are not interchangeable numbers; one is a raw measurement, the other an affinity estimate derived under stated assumptions.

EC50: half-maximal effective concentration

EC50 flips the perspective from inhibition to activation. It is the concentration of an agonist that produces half of its own maximal effect in a functional assay, so it reports potency in generating a response rather than affinity for a binding site. Two compounds can differ sharply in EC50: comparative work on phosphodiesterase inhibitors in isolated tissue reported EC50 values spanning more than an order of magnitude, illustrating how potency is ranked from concentration-response curves (Rump et al., 1994). Importantly, EC50 blends two properties that modern receptor theory treats separately: how well a ligand binds (affinity) and how effectively it activates the receptor once bound (efficacy). Operational models such as the Black-Leff framework were developed precisely to separate these terms, which is why potency alone can be a misleading summary of a molecule’s pharmacology (Kenakin et al., 2011).

Reading these values critically

A few habits make research easier to interpret. Check the units and the log scale, since a tenfold change in Ki or EC50 is a single log unit and easy to underweight. Note whether a number is a binding constant (Ki, Kd) or a functional or operational readout (IC50, EC50), because only the former is meant to travel between assays. Look for the assay conditions and, for IC50 values, whether a Cheng-Prusoff correction was applied. Finally, remember that affinity and efficacy are distinct: contemporary pharmacology increasingly describes ligands by “bias” and pathway-selective efficacy rather than a single potency figure (Kenakin & Strachan, 2018). Treating these numbers as the nuanced, condition-aware measurements they are, rather than universal grades, is the core of evidence literacy in this space.

References

• Cheng H C. The power issue: determination of KB or Ki from IC50. A closer look at the Cheng-Prusoff equation, the Schild plot and related power equations. J Pharmacol Toxicol Methods. 2002. DOI: 10.1016/s1056-8719(02)00166-1
• Vlot A H C, de Witte W E A, Danhof M, et al. Target and Tissue Selectivity Prediction by Integrated Mechanistic Pharmacokinetic-Target Binding and Quantitative Structure Activity Modeling. AAPS J. 2017. DOI: 10.1208/s12248-017-0172-7
• Bentz J, Ellens H. Status of the Structural Mass Action Kinetic Model of P-gp-Mediated Transport Through Confluent Cell Monolayers. Methods Mol Biol. 2021. DOI: 10.1007/978-1-0716-1554-6_27
• Kenakin T, Watson C, Muniz-Medina V, et al. A simple method for quantifying functional selectivity and agonist bias. ACS Chem Neurosci. 2011. DOI: 10.1021/cn200111m
• Kenakin T, Strachan R T. PAM-Antagonists: A Better Way to Block Pathological Receptor Signaling? Trends Pharmacol Sci. 2018. DOI: 10.1016/j.tips.2018.05.001
• Rump A F, Acar D, Klaus W. A quantitative comparison of functional and anti-ischaemic effects of the phosphodiesterase-inhibitors, amrinone, milrinone and levosimendan in rabbit isolated hearts. Br J Pharmacol. 1994. DOI: 10.1111/j.1476-5381.1994.tb13143.x

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Research Use Only. The compounds and concepts discussed here are described strictly for laboratory and scientific research purposes. Nothing on this page is intended for human or animal consumption, nor as medical, diagnostic, or therapeutic advice. Affinity and potency values summarize what has been investigated in preclinical and in vitro research; they are not statements of clinical safety or effect. Always consult primary literature and qualified professionals before drawing conclusions from any research data.