Synergistic metabolic benefits of an exenatide analogue and cholecystokinin in diet-induced obese and leptin-deficient rodents.

AIM: To test the impact of cholecystokinin (CCK) plus either amylin or a glucagon-like peptide-1 receptor (GLP-1R) agonist on metabolic variables in diet-induced obese (DIO) rodents. METHODS: A stabilized acetylated version of CCK-8 (Ac-Y*-CCK-8), selective CCK1 receptor (CCK1R) or CCK2 receptor (CCK2R) agonists, amylin or the GLP-1R agonist and exenatide analogue AC3174 were administered in select combinations via continuous subcutaneous infusion to DIO rats for 14 days, or Lep(ob) /Lep(ob) mice for 28 days, and metabolic variables were assessed. RESULTS: Combined administration of Ac-Y*-CCK-8 with either amylin or AC3174 induced greater than additive weight loss in DIO rats, with the overall magnitude of effect being greater with AC3174 + Ac-Y*-CCK-8 treatment. Co-infusion of AC3174 with a specific CCK1R agonist, but not a CCK2R agonist, recapitulated the weight loss mediated by AC3174 + Ac-Y*-CCK-8 in DIO rats, suggesting that synergy is mediated by CCK1R activation. In a 4 × 4 full-factorial response surface methodology study in DIO rats, a synergistic interaction between AC3174 and the CCK1R-selective agonist on body weight and food intake was noted. Co-administration of AC3174 and the CCK1R-selective agonist to obese diabetic Lep(ob) /Lep(ob) mice elicited a significantly greater reduction in percentage of glycated haemoglobin and food intake relative to the sum effects of monotherapy groups. CONCLUSIONS: The anti-obesity and antidiabetic potential of combined GLP-1R and CCK1R agonism is an approach that warrants further investigation.

Interactions of amylinergic and melanocortinergic systems in the control of food intake and body weight in rodents.

AIMS: Amylinergic and melanocortinergic systems have each been implicated in energy balance regulation. We examined the interactive effects of both systems using gene knockout and pharmacological approaches. METHODS: Acute food consumption was measured in overnight fasted male wild-type (WT) and melanocortin-4 receptor (MC-4R) deficient rats and in male and female WT and amylin knockout mice (AmyKO). Changes in food intake, body weight and composition in male WT and MC-4R deficient rats and in male diet-induced obese (DIO) rats. Pharmacological treatments included either rat amylin, murine leptin and/or the MC-4R agonist, Ac-R[CEH-dF-RWC]-amide. RESULTS: Amylin (10 µg/kg, IP) decreased food intake in WT but not in MC-4R deficient rats (30 and 60 min post-injection). Ac-R[CEH-dF-RWC]-amide (100 µg/kg, IP) suppressed food intake similarly in male WT and AmyKO, but was ineffective in female AmyKO. Amylin (50 µg/kg/day for 28 days) and leptin (125 µg/kg/day) synergistically reduced food intake and body weight in WT and MC-4R deficient rats to a similar extent. Amylin (100 µg/kg) combined with Ac-R[CEH-dF-RWC]-amide (100 µg/kg, IP) decreased acute food intake over 3 h to a greater extent than either agent alone in fasted mice. In DIO rats, additive anorexigenic, weight- and fat-lowering effects were observed over 12 days with the combination of rat amylin (50 µg/kg/day) and Ac-R[CEH-dF-RWC]-amide (2.3 mg/kg, SC injected daily). CONCLUSIONS: Although amylin's acute anorexigenic effects are somewhat blunted in MC-4R deficiency and those of MC-4R agonism in amylin deficiency, these effects are surmountable with pharmacological administration lending therapeutic potential to combined amylin/melanocortin agonism for obesity.

Syntheses of 2-alkylamino- and 2-dialkylamino-4,6-diarylpyridines and 2,4,6-trisubstituted pyrimidines using solid-phase-bound chalcones.

Several substituted 2- and 4-hydroxyacetophenones are linked to Wang resin via a modified Mitsunobu protocol. These resin-bound acetophenones are condensed with aromatic aldehydes, and the resulting chalcones 5 are used for the synthesis of 2-dialkylamino- (9a-d) and 2-alkylamino-4,6-diarylpyridines (11a-f), and 2-alkyl-4,6-diaryl- (14a) and 2,4,6-triarylpyrimidines (14b,c) in a manner suitable for combinatorial applications.

Secondary structure induction in aqueous vs membrane-like environments.

The conformational propensity of the 20 naturally occurring amino acids was determined in aqueous 3-[N-morpholino]propane-sulfonic acid (MOPS) buffer, protein interior-like [nonmicellar sodium dodecylsulfate (SDS)] and membrane-like environments (micellar SDS and lysophosphatidylglycerol/lysophosphatidylcholine micelles) using a single "guest" position in a polyalanine-based model host peptide (Ac-KYA13K-NH2). This model system allows the intrinsic alpha-helical or beta-sheet propensity of the amino acids to be determined without intra- and interchain side chain interactions. The overall environment dependence observed for the conformational propensity for the amino acids studied confirms the importance of determining propensity in lipidic environments to better elucidate the biological functions of proteins. The hydrophobic interactions between peptide side chains and lipids appeared to be the primary forces driving the conformational induction in lipidic environments of the model peptides studied. Finally, when comparing the results of these studies with those reported in the literature, the local environment was found to highly influence 65% of the 20 naturally occurring amino acids.

Polyalanine-based peptides as models for self-associated beta-pleated-sheet complexes.

The occurrence of beta-sheet motifs in a number of neurodegenerative disorders has brought about the need for the de novo design of soluble model beta-sheet complexes. Such model complexes are expected to further the understanding of the interconversion processes that occur from cellular allowed random coil or alpha-helical conformation into insoluble cell-deleterious beta-pleated-sheet motifs. In the present study, polyalanine-based peptides (i.e., derived from Ac-KA14K-NH2) were designed that underwent conformational changes from monomeric random coil conformations into soluble, macromolecular beta-pleated-sheet complexes without any covalent modification. The interconversion was found to be length-, environment-, and concentration-dependent and to be driven by hydrophobic interactions between the methyl groups of the alanine side chains. A series of substitution analogs of Ac-KA14K-NH2 was used to study the amino acid acceptability within the hydrophobic core of the complex, as well as at both termini. The formation of amyloid plaques in a number of amyloidogenic peptides could be related to the presence of amino acids within their sequences that were found to have a high propensity to occur in these model beta-sheet complexes.

Structural characterization and 5'-mononucleotide binding of polyalanine beta-sheet complexes.

A study was initiated into the formation and stability of highly soluble beta-sheet macrostructures. Such beta-sheet macrostructures are useful model systems for the study of the biological function of the hydrophobic core of proteins and for the de novo design of novel catalytic mimics. In the current study, a 16-mer-alanine-based peptide (Ac-KA14K-NH2) that is highly water soluble and adopts an extremely stable macromolecular beta-sheet structure was synthesized. A tyrosine-containing analog (Ac-KYA13K-NH1) was used to study the tertiary structure of the complex by circular dichroism spectroscopy, while the influence of the charges on the complex formation and binding affinity was evaluated using a zwitterionic analog (Ac-KEA13KE-NH1). Both the secondary and tertiary structures of the beta-sheet complex were stable to denaturants, as demonstrated by far- and near-ultraviolet circular dichroism spectroscopy. Binding studies with mononucleotides have shown that the beta-sheet complex binds to molecules through both hydrophobic and electrostatic interactions. These intrinsic properties were found to be a prerequisite for the observed enhanced cleavage of phosphodiester bonds.

Peptide binding domains determined through chemical modification of the side-chain functional groups.

A clear understanding of the specific secondary structure and binding domain resulting from the interactions of proteins and peptides with lipid surfaces will provide insight into the specific functions of biologically active molecules. We have shown in earlier studies that the stationary phases used in reverse-phase high-performance liquid chromatography represent a model artificial lipid surface for the study of induced conformational states of peptides on lipid interaction. We have now used reverse-phase high-performance liquid chromatography to determine the binding domains of peptides and, by extension, of proteins to a lipid surface. This approach consists of performing chemical modifications of specific amino acid side-chain functionalities after the interaction of the peptides with the reverse-phase high-performance liquid chromatography C18 groups. The susceptibility to oxidation was also studied after binding of the same peptides to liposomes. Oxidation of a single methionine residue "walked" through an amphipathic alpha-helical 18-mer peptide was selected to illustrate this approach. The extent of oxidation was found to be clearly dictated by the accessibility of the methionine residue to the aqueous mobile phase. The binding domain found for the peptide in its lipid-induced conformational state was unequivocally the entire hydrophobic face of the amphipathic alpha-helix.

Formation of an extremely stable polyalanine beta-sheet macromolecule.

We have designed a 16-mer peptide composed of a stretch of alanine residues (Ac-KA14K-NH2) which is an effective, simple model for the study of beta-sheet formation in the hydrophobic cores of proteins. This peptide adopts an aqueous soluble "bundling" macromolecular beta-sheet structure, which is extremely stable to a wide range of pHs, temperatures and/or denaturants. Its unusual stability appears to be due to tight hydrophobic packing of the alanine residues in multilayer sheets or micellar forms with the multimeric lysine array being directed outward at the aqueous environment, allowing aqueous solubility.

Stabilization of alpha-helical structures in short peptides via end capping.

The alpha-helix-stabilizing effect of different amino acid residues at the helical termini of short peptides in aqueous solution has been determined. Several dodecapeptides containing alanine, asparagine, aspartate, glutamine, glutamate, and serine at the amino terminus and arginine, lysine, and alanine at the carboxyl terminus were synthesized, and the alpha-helical content of each peptide was measured by using circular dichroism spectroscopy. The trend in alpha-helix-inducing ability of these amino acids was found to be as follows: aspartate > asparagine > serine > glutamate > glutamine > alanine at the amino terminus and arginine > lysine > alanine at the carboxyl terminus. Our results agree with the Presta and Rose hypothesis [Presta, L. G. & Rose, G. D. (1988) Science 240, 1632-1641] on the role of end capping in helix stabilization.