Separation of on-target efficacy from adverse effects through rational design of a bitopic adenosine receptor agonist

Abstract

The concepts of allosteric modulation and biased agonism are revolutionizing modern approaches to drug discovery, particularly in the field of G protein-coupled receptors (GPCRs). Both phenomena exploit topographically distinct binding sites to promote unique GPCR conformations that can lead to different patterns of cellular responsiveness. The adenosine A1 GPCR (A1AR) is a major therapeutic target for cardioprotection, but current agents acting on the receptor are clinically limited for this indication because of on-target bradycardia as a serious adverse effect. In the current study, we have rationally designed a novel A1AR ligand (VCP746)--a hybrid molecule comprising adenosine linked to a positive allosteric modulator--specifically to engender biased signaling at the A1AR. We validate that the interaction of VCP746 with the A1AR is consistent with a bitopic mode of receptor engagement (i.e., concomitant association with orthosteric and allosteric sites) and that the compound displays biased agonism relative to prototypical A1AR ligands. Importantly, we also show that the unique pharmacology of VCP746 is (patho)physiologically relevant, because the compound protects against ischemic insult in native A1AR-expressing cardiomyoblasts and cardiomyocytes but does not affect rat atrial heart rate. Thus, this study provides proof of concept that bitopic ligands can be designed as biased agonists to promote on-target efficacy without on-target side effects.

Fig. 1.

Building biased bitopic agonists. (A) VCP746 is a rationally designed hybrid comprising the endogenous orthosteric agonist (adenosine) linked to a positive allosteric modulator (VCP171); VCP900 is adenosine plus the linker alone. (B) Theoretical behavior of a bitopic agonist. The black circle denotes the orthosteric antagonist (e.g., DPCPX), the blue circle denotes the orthosteric agonist, and the pink circle denotes the allosteric pharmacophore. The scheme on the left represents expectations for a simple competitive interaction, whereas the scheme on the right represents negative allosteric modulation by orthosteric antagonist of allosteric agonist signaling efficacy (as seen for the interaction between DPCPX and VCP171) (Fig. S2). (C) Scheme summarizing ideal bias properties of an A₁AR therapeutic.

Fig. 2.

(A) Pharmacological characterization of hybrid and comparator ligands in whole-cell radioligand binding assays using [³H]DPCPX and membrane-based functional assays of [³⁵S]GTPγS binding in CHO cells stably expressing the human A₁AR. Data represent the mean of three experiments ± SEMs performed in duplicate. (B) The functional interaction between VCP746 and DPCPX conforms to the expectations of a bitopic receptor model (Fig. 1) in assays of A₁AR-mediated ERK1/2 phosphorylation or [³⁵S]GTPγS binding. Data represent the mean of three experiments ± SEMs performed in duplicate.

Fig. 3.

VCP746 is a biased agonist. (A) Effects of selected ligands on A₁AR-mediated inhibition of (Left) forskolin-stimulated cAMP accumulation or (Right) ERK1/2 phosphorylation (pERK1/2) in CHO FlpIn cells stably expressing the human A₁AR. (B) Bias plots showing the effects of equimolar agonist concentrations at the two pathways. (C) Quantification of bias factors using R-PIA as the reference agonist. Data represent the mean of three experiments ± SEMs performed in duplicate. *P < 0.05; Student's t test.

Fig. 4.

VCP746 is cytoprotective but does not cause bradycardia. (A) Effects of the indicated compounds on cell death under conditions of SI in native A₁AR-expressing rat h9c2 caridomyoblasts. Data represent the means ± SEMs of three to nine experiments. (B) Effects of the indicated compounds on cell death under SI conditions in native A₁AR-expressing rat neonatal ventricular cardiomyocytes. Data represent the means ± SEMs of three experiments. (C) Effects of the indicated compounds on rat atrial heart rate (HR). Data represent the means ± SEMs of three experiments. *P < 0.05 as determined by Student's t test (A) or one way ANOVA followed by Dunnett's posttest (B).