9780521516358book.pdf

Quantitative characterisation of competitive antagonists

The ability of a competitive antagonist to reduce the response of a receptor to a given

concentration of agonist can be quantified in two main ways:

• IC 50 value: The antagonist concentration that reduces the response of the receptor,
  in the absence of the antagonist, to a given concentration of agonist by 50%.


• Kbvalue: The dissociation constant for the binding of the antagonist.
  To determine an IC 50 value, the standard procedure is to study the effect of
  increasing concentrations of antagonist on the response to a fixed concentration of
  agonist. In the absence of antagonist the response will be a maximum. As the
  antagonist concentration is increased the response will decrease in a manner that is
  a mirror image of a dose–response curve (Fig. 17.1). From the curve the antagonist
  concentration required to reduce the response by half (IC 50 ) can be determined. If this
  study is repeated for a series of increasing fixed agonist concentrations it will be
  evident that the IC 50 value is critically dependent on the agonist concentration used,
  i.e. it is not an absolute value. In spite of this, it is commonly used, because of its
  simplicity, particularly in the screening of potential therapeutic agents. From knowl-
  edge of the IC 50 value, in principle it is possible to calculate theKbvalue using the
  Cheng–Prusoff equation:

Kb¼

IC 50

ð 1 þ½LŠ=KdÞ ð^17 :^12 Þ

where [L] is the concentration of agonist andKdis its dissociation constant. It is

evident from this equation that IC 50 only approximates toKbwhen [L] is very small

and the denominator approaches 1. Although this equation is commonly used to

calculateKbvalues its application is subject to reservations primarily because inhib-

ition curves do not confirm the nature of the antagonism but also because the

application of the equation is subject to the concentration of agonist used relative

to its EC 50 value. Antagonist equilibrium constants,Kb, are best determined by

application of theSchildt equation:

r¼ 1 þ½BŠ=Kb ð 17 : 13 Þ

or in its logarithmic form:

logðr 1 Þ¼log½BŠlogKb ð 17 : 14 Þ

where [B] is the concentration of antagonist, andris thedose factorthat measures the

amount by which the agonist concentration needs to be increased in the presence of

the antagonist to produce the same response as that obtained in the absence of

antagonist, the assumption being made that the same fraction of receptors needs to

be activated in the presence and absence of the antagonist to produce a given

response. Experimentally, the dose ratio is equal to the dose of agonist required to

give 50% response in the presence of the given antagonist concentration divided by

the EC 50 value.

678 Cell membrane receptors and cell signalling