Graphical representation of the IC50 determination of the inhibition of an enzyme's activity by a small molecule ("drug"). Four different concentrations of the small molecule (ranging from 30 to 300 μM) were tested.

Half maximal inhibitory concentration (IC50) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitory substance (e.g. drug) is needed to inhibit, in vitro, a given biological process or biological component by 50%.[1] The biological component could be an enzyme, cell, cell receptor or microorganism. IC50 values are typically expressed as molar concentration.

IC50 is commonly used as a measure of antagonist drug potency in pharmacological research. IC50 is comparable to other measures of potency, such as EC50 for excitatory drugs. EC50 represents the dose or plasma concentration required for obtaining 50% of a maximum effect in vivo.[1]

IC50 can be determined with functional assays or with competition binding assays.

Sometimes, IC50 values are converted to the pIC50 scale.

Due to the minus sign, higher values of pIC50 indicate exponentially more potent inhibitors. pIC50 is usually given in terms of molar concentration (mol/L, or M), thus requiring IC50 in units of M.[2]

The IC50 terminology is also used for some behavioral measures in vivo, such as the two bottle fluid consumption test. When animals decrease consumption from the drug-laced water bottle, the concentration of the drug that results in a 50% decrease in consumption is considered the IC50 for fluid consumption of that drug.[3]

Functional antagonist assay

The IC50 of a drug can be determined by constructing a dose-response curve and examining the effect of different concentrations of antagonist on reversing agonist activity. IC50 values can be calculated for a given antagonist by determining the concentration needed to inhibit half of the maximum biological response of the agonist.[4] IC50 values can be used to compare the potency of two antagonists.

IC50 values are very dependent on conditions under which they are measured. In general, a higher concentration of inhibitor leads to lowered agonist activity. IC50 value increases as agonist concentration increases. Furthermore, depending on the type of inhibition, other factors may influence IC50 value; for ATP dependent enzymes, IC50 value has an interdependency with concentration of ATP, especially if inhibition is competitive.

IC50 and affinity

Competition binding assays

In this type of assay, a single concentration of radioligand (usually an agonist) is used in every assay tube. The ligand is used at a low concentration, usually at or below its Kd value. The level of specific binding of the radioligand is then determined in the presence of a range of concentrations of other competing non-radioactive compounds (usually antagonists), in order to measure the potency with which they compete for the binding of the radioligand. Competition curves may also be computer-fitted to a logistic function as described under direct fit.

In this situation the IC50 is the concentration of competing ligand which displaces 50% of the specific binding of the radioligand. The IC50 value is converted to an absolute inhibition constant Ki using the Cheng-Prusoff equation formulated by Yung-Chi Cheng and William Prusoff (see Ki).[4][5]

Cheng Prusoff equation

IC50 is not a direct indicator of affinity, although the two can be related at least for competitive agonists and antagonists by the Cheng-Prusoff equation.[6] For enzymatic reactions, this equation is:

where Ki is the binding affinity of the inhibitor, IC50 is the functional strength of the inhibitor, [S] is fixed substrate concentration and Km is the Michaelis constant i.e. concentration of substrate at which enzyme activity is at half maximal (but is frequently confused with substrate affinity for the enzyme, which it is not).

Alternatively, for inhibition constants at cellular receptors:[7]

where [A] is the fixed concentration of agonist and EC50 is the concentration of agonist that results in half maximal activation of the receptor. Whereas the IC50 value for a compound may vary between experiments depending on experimental conditions, (e.g. substrate and enzyme concentrations) the Ki is an absolute value. Ki is the inhibition constant for a drug; the concentration of competing ligand in a competition assay which would occupy 50% of the receptors if no ligand were present.[5]

The Cheng-Prusoff equation produces good estimates at high agonist concentrations, but over- or under-estimates Ki at low agonist concentrations. In these conditions, other analyses have been recommended.[7]

See also

References

  1. 1 2 Hoetelmans RM. "IC50 versus EC50". PK-PD relationships for anti-retroviral drugs. Amsterdam: Slotervaart Hospital. Archived from the original on 2017-05-28 – via U.S. Food and Drug Administration.
  2. ↑ Stewart MJ, Watson ID (July 1983). "Standard units for expressing drug concentrations in biological fluids". British Journal of Clinical Pharmacology. 16 (1): 3–7. doi:10.1111/j.1365-2125.1983.tb02136.x. PMC 1427960. PMID 6882621.
  3. ↑ Robinson SF, Marks MJ, Collins AC (April 1996). "Inbred mouse strains vary in oral self-selection of nicotine". Psychopharmacology. 124 (4): 332–9. doi:10.1007/bf02247438. PMID 8739548. S2CID 19172675.
  4. 1 2 Beck B, Chen YF, Dere W, Devanarayan V, Eastwood BJ, Farmen MW, et al. (November 2017). "Assay Operations for SAR Support". Assay Guidance Manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences. PMID 22553866.
  5. 1 2 "Receptor binding techniques: competition (inhibition or displacement) assays". Pharmacology Guide. Glaxo Wellcome.
  6. ↑ Cheng Y, Prusoff WH (December 1973). "Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction". Biochemical Pharmacology. 22 (23): 3099–108. doi:10.1016/0006-2952(73)90196-2. PMID 4202581.
  7. 1 2 Lazareno S, Birdsall NJ (August 1993). "Estimation of competitive antagonist affinity from functional inhibition curves using the Gaddum, Schild and Cheng-Prusoff equations". British Journal of Pharmacology. 109 (4): 1110–9. doi:10.1111/j.1476-5381.1993.tb13737.x. PMC 2175764. PMID 8401922.
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