Discovery of a novel potent peptide agonist to adiponectin receptor 1

<div><p>Activation of adiponectin receptors (AdipoRs) by its natural ligand, adiponectin has been known to be involved in modulating critical metabolic processes such as glucose metabolism and fatty acid oxidation as demonstrated by a number of <i>in vitro</i> and <i>in vivo</i> studies over last two decades. These findings suggest that AdipoRs’ agonists could be developed into a potential therapeutic agent for metabolic diseases, such as diabetes mellitus, especially for type II diabetes, a long-term metabolic disorder characterized by high blood sugar, insulin resistance, and relative lack of insulin. Because of limitations in production of biologically active adiponectin, adiponectin-mimetic AdipoRs’ agonists have been suggested as alternative ways to expand the opportunity to develop anti-diabetic agents. Based on crystal structure of AdipoR1, we designed AdipoR1’s peptide agonists using protein-peptide docking simulation and screened their receptor binding abilities and biological functions <i>via</i> surface plasmon resonance (SPR) and biological analysis. Three candidate peptides, BHD1028, BHD43, and BHD44 were selected and confirmed to activate AdipoR1-mediated signal pathways. In order to enhance the stability and solubility of peptide agonists, candidate peptides were PEGylated. PEGylated BHD1028 exhibited its biological activity at nano-molar concentration and could be a potential therapeutic agent for the treatment of diabetes. Also, SPR and virtual screening techniques utilized in this study may potentially be applied to other peptide-drug screening processes against membrane receptor proteins.</p></div

Preparation of active AdipoR1.

<p>A. Structural diagram for Adiponectin receptor 1 and its deletion mutant, Δ88. Shaded regions indicate transmembrane domain. ECL1, ECL2, ECL3, and c-terminal domain form extracellular surface. N-terminal 88 residues were deleted in AdipoR1-Δ88. Both constructs have N-terminal flag tag for further purification. B. Over-expressed recombinant AdipoR1 and AdipoR1-Δ88 in insect cells were analyzed before and after anti-flag affinity purification by Western blot with anti-flag antibody. C. AdipoR1-Δ88 purified by anti-flag beads were further purified by size exclusion chromatography to remove other impurities and flag peptide and to replace buffer. Eluents were analyzed by Western blot with anti-flag and anti-AdipoR1 antibodies. D. Purified AdipoR1-Δ88 was analysed by non-reducing SDS-PAGE.</p

Surface plasmon resonance analysis.

<p>ADP355 (A), BHD1028 (B), BHD43 (C), and BHD44 (D) were injected into AdipoR1-Δ88 -immobilized CM5 chip. SPR signals from different concentrations were plotted against concentrations to calculate Kd value.</p

AMPK activation by selected peptides.

<p>A. Differentiated C2C12 myotubes were treated with 0.8, 4, and 20 μM of BHD1028, BHD43, and BHD44. As a positive control, ADP355 and 2.5 μg/ml globular adiponectin were treated. AMPK activation was analyzed by Western blot with anti-pAMPK^thr172, anti-total AMPK, and GAPDH. B. Western blot signals were quantified by densitometer and relative signal were calculated by dividing pAMPK by GAPDH signals.</p

Docking simulation of selected peptides into AdipoR1.

<p>BHD1028 (A), BHD43 (B), and BHD44 (C) were simulated to bind to AdipoR1. Four major binding pockets were indicated with arched lines. Pocket 1 and 2 formed ligand binding site 2 and pocket 3 and 4 formed ligand binding site 1. Expected hydrogen bonds were indicated by red dot lines and key residues were annotated with number.</p

Comparison of selected peptides to adiponectin and ADP355.

<p>Comparison of selected peptides to adiponectin and ADP355.</p

AMPK activation by PEGylated peptides.

<p>A and B. Selected peptides and PEGylated peptides treated to differentiated C2C12 myotubes at 20 μM. C and D. PEGylated BHD1028 induced AMPK and ACC activation with concentration dependent manners. Activation of AMPK and ACC were assessed by Western blot analysis (A and C) and their signals were quantified by densitometer (B and D).</p