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Flexible Modulation of Agonist Efficacy at the Human A3 Adenosine Receptor by the Imidazoquinoline Allosteric Enhancer LUF6000

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Flexible Modulation of Agonist Efficacy at the Human A3 Adenosine Receptor by the Imidazoquinoline Allosteric Enhancer LUF6000

Zhan-Guo Gao et al. BMC Pharmacol.

Abstract

Background: A series of 1H-imidazo- [4,5-c]quinolin-4-amine derivatives, represented by LUF6000 (N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo [4,5-c]quinolin-4-amine), are allosteric modulators of the human A3 adenosine receptor (AR). Here we studied the modulation by LUF6000 of the maximum effect (Emax) of structurally diverse agonists at the A3 AR stably expressed in CHO cells.

Results: In an assay of [35S]GTPgammaS binding, the Emax of the A3 AR agonist Cl-IB-MECA at the A3 AR was lower than that of the non-selective AR agonist NECA. LUF6000 exerted an Emax-enhancing effect at a concentration of 0.1 microM or higher, and was shown to increase the Emax of Cl-IB-MECA and other low-efficacy agonists to a larger extent than that of the high-efficacy agonist NECA. Interestingly, LUF6000 converted a nucleoside A3 AR antagonist MRS542, but not a non-nucleoside antagonist MRS1220, into an agonist. LUF6000 alone did not show any effect. Mathematical modeling was performed to explain the differential effects of LUF6000 on agonists with various Emax. A simple explanation for the observation that LUF6000 has a much stronger effect on Cl-IB-MECA than on NECA derived from the mathematical modeling is that NECA has relatively strong intrinsic efficacy, such that the response is already close to the maximum response. Therefore, LUF6000 cannot enhance Emax much further.

Conclusion: LUF6000 was found to be an allosteric enhancer of Emax of structurally diverse agonists at the A3 AR, being more effective for low-Emax agonists than for high-Emax agonists. LUF6000 was demonstrated to convert an antagonist into an agonist, which represents the first example in G protein-coupled receptors. The observations from the present study are consistent with that predicted by mathematical modeling.

Figures

Figure 1
Figure 1
Chemical structures of the agonists, antagonists, and an allosteric modulator of the A3 adenosine receptor used in the present study.
Figure 2
Figure 2
Effect of LUF6000 on agonist-induced activation of the human A3 AR studied with a [35S]GTPγS binding assay. Incubations were started by addition of the membrane suspension (5 μg protein/tube) to the test tubes, and carried out in duplicate for 30 min at 25°C, except for B and C in which LUF6000 or both LUF6000 and an agonist were incubated for 20 min with membranes and other components before the addition of [35S]GTPγS (final concentration 0.2 nM). The experimental procedures are described in the Materials and Methods section. Results were from 3–5 independent experiments performed in duplicate. The basal values typically ranged from 800 to 1200 cpm. The maximal values are typically from 2000 to 2500 cpm. A. Cl-IB-MECA. B. Cl-IB-MECA (LUF6000 was incubated with membranes 20 min before the addition of other components). C. Cl-IB-MECA (Both LUF6000 and Cl-IB-MECA were incubated 20 min with membranes before the addition of other ingredients); D. NECA; E. MRS541; MRS542. The maximum stimulation of NECA in the absence of enhancers was expressed as 100%.
Figure 3
Figure 3
Effect of LUF6000 on various human A3 AR ligands, studied with a [35S]GTPγS binding assay. Incubations were started by addition of the membrane suspension (5 μg protein/tube) to the test tubes, and carried out in duplicate for 30 min at 25°C. The experimental procedures are described in the Materials and Methods section. Results were from 3–5 independent experiments performed in duplicate. The basal values typically ranged from 800 to 1200 cpm. The maximal values are typically from 2000 to 2500 cpm.
Figure 4
Figure 4
Effect of LUF6000 on agonist-induced activation of the human A1 AR studied with a [35S]GTPγS binding assay. Incubations were started by addition of the membrane suspension (5 μg protein/tube) to the test tubes, and carried out in duplicate for 30 min at 25°C. Results were from 3 independent experiments performed in duplicate. The basal values ranged from 700 to 1000 cpm. The maximal values are typically from 1500 to 1800 cpm.
Figure 5
Figure 5
Simulation of concentration-effect curves with MatLab. The equations from Hall (2000) were used to derive conditions that vary in efficacy and potency. The parameters in these equations are explained and discussed in the text. Parts A, B, C and D mimic the experimental concentration-effect curves of Figures 2D, 2C, 2E and 2F, respectively.

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References

    1. Fredholm BB, IJzerman AP, Jacobson KA, Klotz KN, Linden J. International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacol Rev. 2001;53:527–552. - PubMed
    1. Chen Y, Corriden R, Inoue Y, Yip L, Hashiguchi N, Zinkernagel A, Nizet V, Insel PA, Junger WG. ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors. Science. 2006;314:1792–1795. doi: 10.1126/science.1132559. - DOI - PubMed
    1. Jacobson KA, Gao ZG. Adenosine receptors as therapeutic targets. Nat Rev Drug Disc. 2006;5:247–264. doi: 10.1038/nrd1983. - DOI - PMC - PubMed
    1. Yan L, Burbiel JC, Maass A, Müller CE. Adenosine receptor agonists: from basic medicinal chemistry to clinical development. Expert Opin Emerg Drugs. 2003;8:537–576. doi: 10.1517/14728214.8.2.537. - DOI - PubMed
    1. Gao ZG, Jacobson KA. Emerging adenosine receptor agonists. Expert Opin Emerging Drugs. 2007;12:479–492. doi: 10.1517/14728214.12.3.479. - DOI - PubMed

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