ABSTRACT
Ghrelin and Y2 receptors play a central role in appetite regulation inducing opposite effects. The Y2 receptor induces satiety, while the ghrelin receptor promotes hunger and weight gain. However, the food regulating system is tightly controlled by interconnected pathways where redundancies can lead to poor efficacy and drug tolerance when addressing a single molecule. We developed a multitarget strategy to synthesize dual peptides simultaneously inhibiting the ghrelin receptor and stimulating the Y2 receptor. Dual peptides showed dual activity in vitro, and one compound induced a slight diminution of food intake in a rodent model of obesity. In addition, stability studies in rats revealed different behaviors between the dual peptide and its corresponding monomers. The Y2 receptor agonist was unstable in blood, while the dual peptide showed an intermediate stability compared to that of the highly stable ghrelin receptor inverse agonist.
Subject(s)
Anti-Obesity Agents/pharmacology , Body Weight/drug effects , Eating/drug effects , Ghrelin , Peptides/chemistry , Peptides/pharmacology , Receptors, Gastrointestinal Hormone/agonists , Receptors, Ghrelin/antagonists & inhibitors , Amino Acid Sequence , Animals , Anti-Obesity Agents/chemistry , Binding, Competitive , COS Cells , Chlorocebus aethiops , Drug Design , Female , Humans , Mice, Inbred C57BL , Molecular Sequence Data , Molecular Targeted Therapy , Peptides/chemical synthesis , Receptors, Ghrelin/agonistsABSTRACT
Obesity is characterized by an excessive accumulation of body fat, for which impaired adipogenesis is believed to play a crucial role. As a gatekeeper of early adipogenesis, protein delta homolog 1 (DLK1) has a pivotal role in deciding whether pre-adipocytes will differentiate, determining the balance between healthy and unhealthy fat tissue. Here, an expression system for the cysteine-rich soluble human DLK1 was established. DLK1 was overexpressed in Escherichia coli BL21(DE3)pLysRARE, purified by affinity chromatography and refolded by stepwise dialysis. Identity, purity, secondary structure and refolding efficiency were determined. Proteolytic digestion followed by mass spectrometry analysis proved correct disulfide bridge formation. The biological activity of DLK1 was examined by differentiation assays in murine pre-adipocyte-like 3T3-L1 cells. Thereby, recombinantly produced DLK1 was shown to inhibit adipogenesis in a concentration- and time-dependent manner. All in all, our approach gives access to large amounts of active DLK1 and can be transferred to related proteins.
Subject(s)
Adipocytes/drug effects , Inclusion Bodies/chemistry , Intercellular Signaling Peptides and Proteins/isolation & purification , Membrane Proteins/isolation & purification , Plasmids/metabolism , Recombinant Proteins/isolation & purification , 3T3-L1 Cells , Adipocytes/cytology , Adipocytes/metabolism , Adipogenesis/genetics , Animals , Calcium-Binding Proteins , Cell Differentiation/drug effects , Chromatography, Affinity , Cloning, Molecular , Disulfides/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/pharmacology , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Mice , Plasmids/chemistry , Protein Refolding , Protein Stability , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Solubility , Urea/chemistryABSTRACT
The ghrelin receptor displays a high constitutive activity suggested to be involved in the regulation of appetite and food intake. Here, we have created peptides with small changes in the core binding motif -wFw- of the hexapeptide KwFwLL-NH(2) that can swap the peptide behavior from inverse agonism to agonism, indicating the importance of this sequence. Introduction of ß-(3-benzothienyl)-d-alanine (d-Bth), 3,3-diphenyl-d-alanine (d-Dip) and 1-naphthyl-d-alanine (d-1-Nal) at position 2 resulted in highly potent and efficient inverse agonists, whereas the substitution of d-tryptophane at position 4 with 1-naphthyl-d-alanine (d-1-Nal) and 2-naphthyl-d-alanine (d-2-Nal) induces agonism in functional assays. Competitive binding studies showed a high affinity of the inverse agonist K-(d-1-Nal)-FwLL-NH(2) at the ghrelin receptor. Moreover, mutagenesis studies of the receptor revealed key positions for the switch between inverse agonist and agonist response. Hence, only minor changes in the peptide sequence can decide between agonism and inverse agonism and have a major impact on the biological activity.
