Received for publication May 4, 2006.
Revised July 18, 2006.
Accepted for publication July 19, 2006.

Determining the Potency and Molecular Mechanism of Action of Insurmountable Antagonists

Abstract

Insurmountable antagonism (maximal response to the agonist depressed) can result from a temporal inequilibrium involving a slow offset orthosteric antagonist or be the result of an allosteric modulation of the receptor. The former mechanism is operative when the antagonist, agonist and receptors cannot come to proper equilibrium during the time allotted for collection of agonist response (hemi-equilibrium conditions). Allosteric effects (changes in the conformation of the receptor through binding of the allosteric modulator to a separate site) can preclude the agonist-induced production of response leading to depression of maximal responses. In these cases effects on receptor affinity can be observed as well. The first premise of this paper is that system independent estimates of insurmountable antagonist potency can be made with no prior knowledge of molecular mechanism through the use of pA₂ (-log molar concentration of antagonist producing a 2-fold shift of the concentration response curve) measurements The relationship between the pA₂ and antagonist pK_B (-log equilibrium dissociation constant of the antagonist-receptor complex) is described; the former is an extremely close approximation of the latter in most cases. The second premise is that specially designed experiments are required differentiate orthosteric vs allosteric mechanisms; simply fitting of data to orthosteric or allosteric theoretical models can lead to ambiguous results. A strategy to determine whether the observed antagonism is orthosteric (agonist and antagonist competing for the same binding site on the receptor) or allosteric in nature, is described which involves the detection of the hallmarks of allosteric response, namely saturation and probe dependence of effect.

Key words: Allosteric Antagonism, Drug discovery, Hemi-Equilibrium States, Non-Competitive Antagonism, Quantifying potency of Antagonists, Receptor Theory

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