Abstract
BackgroundA 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.ResultsIn an assay of [35S]GTPγS 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 μM 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.ConclusionLUF6000 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.
Highlights
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)
We extended our previous observations by studying the nature of the potentially flexible modulation by LUF6000 of the agonists with a selection of A3 AR agonists having a distribution of Emax values in A3 AR-expressing Chinese hamster ovary (CHO) cells using a [35S]GTPγS binding assay [18]
The allosteric modulator (Figure 1) used in the present study is the imidazoquinoline derivative LUF6000, which has been shown to retard agonist radioligand dissociation and to increase agonist Emax, as demonstrated using a cyclic AMP functional assay [17]
Summary
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). Subtype-selective AR agonists have been developed, the selectivity for some organs or tissues is nearly unachievable using orthosteric agonists that act directly at the principal ligand binding site of the receptor. This is due to the wide distribution of ARs and, a number of agonists were discontinued after the initial phases of clinical trials [3,4,5]. An advantage of an allosteric enhancer of a GPCR over its native, orthosteric activator is that greater selectivity can be achieved. The effect of an endogenous agonist, which may be insufficient in a particular disease state, may be magnified in a temporally and spatially specific manner through allosteric modulation
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