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Alternative approach to the Schild plot analysis
2135 P.fr.O19 [
Alternative approach to the Schild plot analysis P~ch, G., Brunner, F. and Kiihberger, E. Institute of Pharmacodynamics and Toxicology, University of Graz. Universitiitsplatz 2, A-8010 Graz, Austria
ConventionaHy, the affinity constant of a competitive antagonist (K e) is derived from the shift of an agonist dose response curve (DRC) as a function of antagonist concentration B. The shift of the agonist EDso is expressed by the dose ratio DR and antagonist affinity is calculated as K B = B/(DR-I). The use of several antagonist concentrations and plotting log (DR-I) vs log B yields - l o g K e = pA 2. If this Schi!d regression is linear and has a slope of unity, a competitive antagonism is inferred. Theory: We have devised an alternative approach by quantifying the extent of antagonism at a given agonist concentration A according to the relationship: K B = B~/(DR-1). B~ equals that antagonist concentration which reduces any agonist effect between the EDs0 and Emax to E~.~/2, and DR is the dose ratio calculated for the chosen agonist concentration A (i.e., in the presence of an ant~,gonist) relative to the respective agonist control DRC (i. e., in the absence of antagonist). To obtain B,, the antagonist effect is plotted as a function of antagonist concentration yielding an antagonist DRC from which the suitable measure of antagonist potency, i.e. ICso may be derived. ICso of the antagonist equals Bx at Emax of the agonist, but B~ < ICs0 at submaximal effective concentrations of the agonist. If the interaction between agonist and antagonist is competitive, K B should be independent of the agonist concentration A chosen. The use of several agonist concentrations A, A', A " . . . allows the calculation of the respective B~ values and DRA-1, DRA-1, D R y . . . which may be regressed in analogy to the classical Schild equation to yield an alternative PA2-value.Results: This new approach to estimate antagonist affinity via "alternative pA2-analysis" was tested with data from our own laboratory (negative inotropic effect of bovine trabecular heart muscle) as well as published agonist DRCs (Ari6ns et al., 1956; Kenakin, 1982). The following competitive antagonists were used: Pirenzepine and 4-DAMP (muscarinic receptor); BuNMe3Br (nicotinic receptor), and butoxamine (/~-adrenergic receptor). Since B~ values showed greater variability when calculated from antagonist DRCs at DR < 3 than at DR > 3, we used only B~ values obtained at DR > 3. Comparison of alternative and classical pA2-values gave an excellent agreement in the observed range of pA 2 = 8.37-5.71 (N = 7; slope,= 0.996; r--0.999). Thus, the alternative approach yielded the same pA2-values as the conventional procedure. Discussion: The "alternative" Schild plot appears of advantage where the supply and/or the solubility of the agonist is limited. Agonist DRCs in the presence of higher concentrations of antagonist need not, reach the maximum effect. Yet, they can yield complete DRCs of antagonist effects (since the latter are derived by vertical analysis of agonist DRCs) and thus still provide a precise estimate of pA 2. References Ariens, E.J. et al., 1956, Arzneim.-Forsch. (Drug Res.) 6, 282. Kenaldn, T.P., 1982, J. Pharmacol. Exp. Ther. 223, 416. I P.fr.020 [
Spectral map analysis of receptor amino acid sequences Moereels, H., Lewi, P.J. and Leysen, J.E. Janssen Research Foundation, B-2340 Beerse, Belgium
Today, with the introduction of cloning techniques, we entered a new era in the field of receptorology. The cloned membrane receptors, coupled to guanine nucleotide binding proteins (G-proteins), provide us with the amino acid
Alternative approach to the Schild plot analysis P~ch, G., Brunner, F. and Kiihberger, E. Institute of Pharmacodynamics and Toxicology, University of Graz. Universitiitsplatz 2, A-8010 Graz, Austria
ConventionaHy, the affinity constant of a competitive antagonist (K e) is derived from the shift of an agonist dose response curve (DRC) as a function of antagonist concentration B. The shift of the agonist EDso is expressed by the dose ratio DR and antagonist affinity is calculated as K B = B/(DR-I). The use of several antagonist concentrations and plotting log (DR-I) vs log B yields - l o g K e = pA 2. If this Schi!d regression is linear and has a slope of unity, a competitive antagonism is inferred. Theory: We have devised an alternative approach by quantifying the extent of antagonism at a given agonist concentration A according to the relationship: K B = B~/(DR-1). B~ equals that antagonist concentration which reduces any agonist effect between the EDs0 and Emax to E~.~/2, and DR is the dose ratio calculated for the chosen agonist concentration A (i.e., in the presence of an ant~,gonist) relative to the respective agonist control DRC (i. e., in the absence of antagonist). To obtain B,, the antagonist effect is plotted as a function of antagonist concentration yielding an antagonist DRC from which the suitable measure of antagonist potency, i.e. ICso may be derived. ICso of the antagonist equals Bx at Emax of the agonist, but B~ < ICs0 at submaximal effective concentrations of the agonist. If the interaction between agonist and antagonist is competitive, K B should be independent of the agonist concentration A chosen. The use of several agonist concentrations A, A', A " . . . allows the calculation of the respective B~ values and DRA-1, DRA-1, D R y . . . which may be regressed in analogy to the classical Schild equation to yield an alternative PA2-value.Results: This new approach to estimate antagonist affinity via "alternative pA2-analysis" was tested with data from our own laboratory (negative inotropic effect of bovine trabecular heart muscle) as well as published agonist DRCs (Ari6ns et al., 1956; Kenakin, 1982). The following competitive antagonists were used: Pirenzepine and 4-DAMP (muscarinic receptor); BuNMe3Br (nicotinic receptor), and butoxamine (/~-adrenergic receptor). Since B~ values showed greater variability when calculated from antagonist DRCs at DR < 3 than at DR > 3, we used only B~ values obtained at DR > 3. Comparison of alternative and classical pA2-values gave an excellent agreement in the observed range of pA 2 = 8.37-5.71 (N = 7; slope,= 0.996; r--0.999). Thus, the alternative approach yielded the same pA2-values as the conventional procedure. Discussion: The "alternative" Schild plot appears of advantage where the supply and/or the solubility of the agonist is limited. Agonist DRCs in the presence of higher concentrations of antagonist need not, reach the maximum effect. Yet, they can yield complete DRCs of antagonist effects (since the latter are derived by vertical analysis of agonist DRCs) and thus still provide a precise estimate of pA 2. References Ariens, E.J. et al., 1956, Arzneim.-Forsch. (Drug Res.) 6, 282. Kenaldn, T.P., 1982, J. Pharmacol. Exp. Ther. 223, 416. I P.fr.020 [
Spectral map analysis of receptor amino acid sequences Moereels, H., Lewi, P.J. and Leysen, J.E. Janssen Research Foundation, B-2340 Beerse, Belgium
Today, with the introduction of cloning techniques, we entered a new era in the field of receptorology. The cloned membrane receptors, coupled to guanine nucleotide binding proteins (G-proteins), provide us with the amino acid