IC50 → Ki converter

What this calculator does

An IC50 is the inhibitor concentration that halves the measured signal under your particular assay conditions. It changes with substrate concentration, tracer concentration and enzyme concentration, so IC50 values from different labs or different assay formats cannot be compared directly. The inhibition constant K_i is an equilibrium dissociation constant for the enzyme–inhibitor complex, and in principle it is independent of the assay. Converting IC50 to K_i is how you put compounds measured under different conditions onto a common scale. It is also needed before turning potency into a binding free energy with the Kd ↔ ΔG converter.

Which conversion is correct depends on the mechanism. That is the part most often got wrong: the familiar Cheng–Prusoff equation applies only to competitive inhibition. This tool covers the common cases:

Worked example

A kinase inhibitor gives IC50 = 100 nM in an assay run with ATP at its K_m (10 µM), and it is ATP-competitive. Then [S]/K_m = 1 and

Ki = 100 nM / (1 + 10/10) = 50 nM, so pK_i = 7.30.

The same compound tested at a physiological ATP concentration of 1 mM (100 × K_m) would show IC50 ≈ 100 × 50 nM ≈ 5 µM, even though its affinity has not changed. This is why biochemical and cellular kinase potencies often differ by one to two orders of magnitude. The calculator warns when [S]/K_m is above 10, because the result then becomes very sensitive to errors in K_m.

Second example, a radioligand assay: IC50 = 30 nM, radioligand at 1 nM with K_d = 0.5 nM. K_i = 30 / (1 + 1/0.5) = 10 nM. Select "Competition binding" above to reproduce it.

Assumptions and when the conversion breaks

• Rapid equilibrium and steady state. Cheng–Prusoff assumes the inhibitor, substrate and enzyme reach equilibrium and that the initial-velocity conditions hold. Slow-binding or covalent inhibitors (time-dependent IC50) need k_inact/K_I analysis instead.
• Free equals total inhibitor. If the enzyme concentration is not much lower than the IC50, part of the inhibitor is bound and depleted, and the measured IC50 has a floor of about [E]/2 (tight binding). If you enter [E], the tool applies the first-order correction IC50 − [E]/2 when IC50 < 10·[E], and rejects IC50 values at or below [E]/2. For really tight binders, fit the Morrison equation to the full dose–response curve.
• One site, Hill slope ≈ 1. A Hill slope far from 1 (outside about 0.7–1.3) suggests aggregation, multiple sites, non-specific inhibition or assay artefacts. The conversion is then questionable, and the tool flags it.
• Correct K_m and K_d. These must have been measured under the same buffer, temperature and enzyme construct as the IC50. A twofold error in K_m at [S] = 10·K_m gives almost a twofold error in K_i.
• Known mechanism. If you have not determined the mechanism (for example by varying [S]), the "competitive" result is an assumption, and you should say so when you report it.

Reporting tips

State the equation used, the [S] (or [L]) and K_m (or K_d) values, and whether a tight-binding correction was applied. When comparing compounds, work with pK_i, because errors in potency are roughly log-normal. If you plan to compare K_i values with docking scores or MM-PBSA energies, read the MM-PBSA tutorial first: end-point methods usually rank compounds better than they predict absolute ΔG.

References

1. Cheng Y, Prusoff WH. Relationship between the inhibition constant (K_I) and the concentration of inhibitor which causes 50 per cent inhibition (I₅₀) of an enzymatic reaction. Biochem Pharmacol 1973;22:3099–3108. doi:10.1016/0006-2952(73)90196-2
2. Copeland RA. Evaluation of Enzyme Inhibitors in Drug Discovery, 2nd ed. Wiley, 2013. doi:10.1002/9781118540398
3. Cer RZ, Mudunuri U, Stephens R, Lebeda FJ. IC50-to-Ki: a web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding. Nucleic Acids Res 2009;37:W441–W445. doi:10.1093/nar/gkp253
4. Morrison JF. Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim Biophys Acta 1969;185:269–286. doi:10.1016/0005-2744(69)90420-3
5. Copeland RA, Lombardo D, Giannaras J, Decicco CP. Estimating K_I values for tight binding inhibitors from dose-response plots. Bioorg Med Chem Lett 1995;5:1947–1952. doi:10.1016/0960-894X(95)00330-V

Results are for research and education and are not a substitute for checking against the primary literature. See the disclaimer. Found a bug? Report it.