doi: 10.1021/acs.jmedchem.8b00238. Epub 2018 May 9.
Developing a Biased Unmatched Bivalent Ligand (BUmBL) Design Strategy to Target the GPCR Homodimer Allosteric Signaling (cAMP over β-Arrestin 2 Recruitment) Within the Melanocortin Receptors
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- PMID:29669202
- PMCID: PMC6226371
- DOI: 10.1021/acs.jmedchem.8b00238
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Developing a Biased Unmatched Bivalent Ligand (BUmBL) Design Strategy to Target the GPCR Homodimer Allosteric Signaling (cAMP over β-Arrestin 2 Recruitment) Within the Melanocortin Receptors
Cody J Lensing et al. J Med Chem..
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Abstract
Understanding the functional relevance of G protein-coupled receptor (GPCR) homodimerization has been limited by the insufficient tools to assess asymmetric signaling occurring within dimers comprised of the same receptor type. We present unmatched bivalent ligands (UmBLs) to study the asymmetric function of melanocortin homodimers. UmBLs contain one agonist and one antagonist pharmacophore designed to target a melanocortin homodimer such that one receptor is occupied by an agonist and the other receptor by an antagonist pharmacophore. First-in-class biased UmBLs (BUmBLs) targeting the human melanocortin-4 receptor (hMC4R) were discovered. The BUmBLs displayed biased agonism by potently stimulating cAMP signaling (EC50 ∼ 2-6 nM) but minimally activating the β-arrestin recruitment pathway (≤55% maximum signal at 10 μM). To our knowledge, we report the first single-compound strategy to pharmacologically target melanocortin receptor allosteric signaling that occurs between homodimers that can be applied straightforwardly in vitro and in vivo to other GPCR systems.
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Figures
Hypothesized interaction of ligands with asymmetrically signaling melanocortin homodimers. A) Monovalent agonist ligands (blue circle) could occupy both receptors and result in both cAMP signaling and β-arrestin recruitment. B) Agonist homobivalent ligands (blue circle connected with black linker) could result in similar functional cAMP assays as monomeric ligands in spite of increased binding affinities due to asymmetric signaling. C) The working paradigm herein in which biased unmatched bivalent ligands (BUmBLs) containing an agonist pharmacophore (blue circle) and antagonist pharmacophore (red octagon) are postulated to result in biased signaling by agonizing one signaling pathway while antagonizing the other pathway when bound to the asymmetrically signaling homodimer.
Illustrations of thein vitro functional pharmacology of MUmBLs at the MC4R.A) The cAMP signaling potency at the hMC4R was determined by AlphaScreen® assays.B) andC) The β-arrestin recruitment potency at the hMC4R was determined by PRESTO-Tango assays. The
symbol represents the two monovalent tetrapeptides Ac-His-DPhe-Arg-Trp-NH2 and Ac-His-DNal(2’)-Arg-Trp-NH2 assayed together each at the indicated M concentration such that pharmacophore concentration is the same as the bivalent pharmacophore concentration. Functional cAMP data was normalized as discussed in experimental section to show tradition dose response curve with increasing response at increasing agonist concentrations.D) Ligand induced response on bioluminescence resonance energy transfer (BRET) signal using the mMC4R-NanoLuc and mMC4R-HaloTag homodimer. Maximal BRET signal (100%) was defined as the signal measured when assay buffer (represented as A) was added. Each ligand was dosed at 10−5, 10−7 and 10−9 M. Significance was determined using a one-way ANOVA to determine overall significance upon treatment followed by a Bonferroni post-hoc test to compare each ligand concentration to assay buffer control (A). * p<0.05, ** p<0.01, *** p<0.001. Data shown as the mean ± standard error of the mean (SEM) determined from three independent experiments.
symbol represents the two monovalent tetrapeptides Ac-His-DPhe-Arg-Trp-NH2 and Ac-His-DNal(2’)-Arg-Trp-NH2 assayed together each at the indicated M concentration such that pharmacophore concentration is the same as the bivalent pharmacophore concentration. Functional cAMP data was normalized as discussed in experimental section to show tradition dose response curve with increasing response at increasing agonist concentrations.D) Ligand induced response on bioluminescence resonance energy transfer (BRET) signal using the mMC4R-NanoLuc and mMC4R-HaloTag homodimer. Maximal BRET signal (100%) was defined as the signal measured when assay buffer (represented as A) was added. Each ligand was dosed at 10−5, 10−7 and 10−9 M. Significance was determined using a one-way ANOVA to determine overall significance upon treatment followed by a Bonferroni post-hoc test to compare each ligand concentration to assay buffer control (A). * p<0.05, ** p<0.01, *** p<0.001. Data shown as the mean ± standard error of the mean (SEM) determined from three independent experiments.
Illustrations of a previously reported model for allosteric interactions in GPCR dimers. (Durroux, 2005; Casado et al., 2007). In this model, GPCRs oscillate through different conformational states. Different conformations have different propensity to signal through cAMP or through β-arrestin. Signaling is represented by green arrows (B, C, E, F, H, I, L). Conformational changes are represented based on receptor highlighting (B, C, D, E, F, H, I, L). The binding of an agonist pharmacophore to one receptor that signals through cAMP stabilizes the second receptor’s conformation to increases its propensity to signal through the β-arrestin recruitment pathway (State E). Therefore, the second agonist binding event results in β-arrestin recruitment (State F). The BUmBL design strategy can be used to block the β-arrestin recruitment by increasing the likelihood of an antagonist pharmacophore binding the second receptor in the homodimer (States G–I). Even if the opposite binding order occurs, the antagonist blocks β-arrestin recruitment since it is already bound to the receptor after the agonist induces a conformational change (States J–L). This model assumes that the receptors are dimeric in nature, but they are likely in an equilibrium as monomers and higher-order oligomers. This models also assumes that the bivalent synergistic binding mode is favored with MUmBLs due to the decreased entropic cost of binding of the second pharmacophore. It is possible that MUmBLs compete in monovalent fashion (Supplemental Fig. 1), but then the increased binding affinity observed with bivalent ligands would not be expected.
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