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, 59 (7), 3112-28

An in Vitro and in Vivo Investigation of Bivalent Ligands That Display Preferential Binding and Functional Activity for Different Melanocortin Receptor Homodimers

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An in Vitro and in Vivo Investigation of Bivalent Ligands That Display Preferential Binding and Functional Activity for Different Melanocortin Receptor Homodimers

Cody J Lensing et al. J Med Chem.

Abstract

Pharmacological probes for the melanocortin receptors have been utilized for studying various disease states including cancer, sexual function disorders, Alzheimer's disease, social disorders, cachexia, and obesity. This study focused on the design and synthesis of bivalent ligands to target melanocortin receptor homodimers. Lead ligands increased binding affinity by 14- to 25-fold and increased cAMP signaling potency by 3- to 5-fold compared to their monovalent counterparts. Unexpectedly, different bivalent ligands showed preferences for particular melanocortin receptor subtypes depending on the linker that connected the binding scaffolds, suggesting structural differences between the various dimer subtypes. Homobivalent compound 12 possessed a functional profile that was unique from its monovalent counterpart providing evidence of the discrete effects of bivalent ligands. Lead compound 7 significantly decreased feeding in mice after intracerebroventricular administration. To the best of our knowledge, this is the first report of a melanocortin bivalent ligand's in vivo physiological effects.

Figures

Figure 1
Figure 1
Design of ligands from selected scaffolds and linkers.
Figure 2
Figure 2
Crude RP-HPLC analytical chromatograms at 214 nm of 3 (mass of 961.6) in a gradient from 10% to 90% MeCN or MeOH in water containing 0.1 % trifluoroacetic acid at a flow rate of 1.5 mL/min over 35 minutes (5 to 20 minutes are shown) using an analytical Vydac C18 column (Vydac 218TP104). (A) Analytical HPLC trace in MeCN of crude peptide 3 after a three hour cleavage which shows only one major peak. A major impurity peak (mass of 685.4) is masked in this chromatogram. (B) Analytical HPLC trace in MeOH of crude peptide 3 after a three hour cleavage which identifies both the desired product and an impurity peak masked in MeCN chromatogram. (C) Co-injection of crude 3 from three hour cleavage with purified 1 (mass of 685.4) increases the intensity of the impurity peak demonstrating similar retention times. (D) A shorter cleavage time of 1.5 hours diminishes degradation product giving better crude peptide purity.
Figure 3
Figure 3
Illustrations of the competitive binding experiments at the mMC1R, mMC3R, and mMC4R. Top figures shows the formula image based ligands. The bottom figures show the formula image based ligands.
Figure 4
Figure 4
Proposed binding mode of the bivalent ligands. (A) First pharmacophore engages GPCR dimer or two neighboring binding sites. (B) The first binding event tethers the second pharmacophore in close proximity to the second binding site significantly increasing the likelihood of the second binding event. (C) The second pharmacophore binds with low entropic cost.
Figure 5
Figure 5
Postulated rationale for linker-dependent preferences at the different melanocortin homodimer subtypes. The different linker systems had varying effects on enhancing binding or functional responses depending on which receptor subtype was expressed. Since the linkers connect the same pharmacophore, it appears the difference are due to the linkers' physicochemical properties such as linker length. These differences suggest that there are differences between the various subtypes of melanocortin receptor dimers such as the distance between tandem binding sites (see text). The figure demonstrates how different distances between tandem binding sites would show preference for the different length linker systems.
Figure 6
Figure 6
Illustrations of the in vitro functional pharmacology at the mMC1R, mMC3R, mMC4R, and mMC5R of the formula image based ligands. Top figures show the bivalent ligands compared to the control peptide 1. The bottom figures show the effects of the linkers plus pharmacophore compared to control peptide 1.
Figure 7
Figure 7
Illustrations of the in vitro functional agonist pharmacology at the mMC1R, mMC3R, mMC4R, and mMC5R of the formula image based ligands.
Figure 8
Figure 8
Correlation of IC50 (nM) vs EC50 (nM) at the different receptor subtypes for formula image based ligands. The mMC4R had a relatively linear correlation between receptor activation and ligand binding. At the mMC1R there appears to be relatively little correlation. The lack of correlation stresses the importance of studying ligands' binding affinity and functional effects in complementary assays. Data is shown as mean ± SEM.
Figure 9
Figure 9
Cumulative food intake following intracerebroventricular administration of either saline (n=16) or 7 in saline (n=8) in male wild type mice. Data is shown as mean ± SEM. Data was analyzed using the PRISM program (v4.0; GraphPad Inc.) by a one-way ANOVA followed by a Bonferroni post test in order to compare individual doses to saline administration. *p<0.05, ** p=0.01.
Scheme 1
Scheme 1. Synthesis of bivalent ligands and control ligands
Synthetic scheme for synthesis of bivalent ligands and control ligands. (A) The synthesis of N-terminal linker controls and bivalent ligands by microwave synthesis. (B) Synthesis of C-terminal linker controls using a semi-automated synthesizer. (a) 20 % piperidine in DMF (b) Fmoc-NH-AA-COOH or Fmoc-NH-PEDG20-COOH, HBTU, DIEA in DMF. Repeat (a) and (b) to achieve desired sequence. When Fmoc-NH-PEDG20-COOH was incorporated, reaction was allowed to proceed for an extra hour at room temperature. (c) 75% acetic anhydride/ 25% pyridine. (d) Cleavage with 91% TFA, 3% EDT, 3% TIS, 3% water for 1.5-3 hours.

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