Synthesis of a possible glucagon inhibitor by semisynthesis of a glucagon analogue of [HARG¹²]-glucagon and [DES-HIS¹][HARG¹²]-glucagon
PublisherThe University of Arizona.
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Degree GrantorUniversity of Arizona
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Design and synthesis of conformationally constrained glucagon analogues to study the conformational features important for glucagon bioactivity.Lin, Ying.; Hruby, Victor J.; Bates, Robert B.; Mash, Eugene A., Jr.; Law, John H.; Hadley, Mac E. (The University of Arizona., 1993)We have synthesized ten glucagon analogues that are either conformationally constrained systematically in the middle portion of the molecule, or modified from the known superagonist analogue [Lys¹⁷,¹⁸, Glu²¹]glucagon to study the structure-activity relationships of glucagon. The analogues were prepared using the solid-phase peptide synthesis method. Cyclizations were accomplished by forming the side chain lactam (amide) bridges on the resin. All peptide analogues were cleaved from the solid support, deprotected by the low-high HF procedure, and purified by a combination of gel filtration chromatography and dialysis followed by reverse-phase high performance liquid chromatography. A new characterization method for cyclic glucagon analogues using fast atom bombardment mass spectrometry with endoproteinase Asp-N peptide mapping has been developed that has provided unequivocal confirmation of the presence and site of the rings as well as the amino acid compositions. Receptor binding and adenylate cyclase activity assays and circular dichroism spectroscopy have been used to reveal the role of the structure and conformation of the middle portion of the molecule. The effects of the modification of the 17, 18 and 21 positions on the superagonist activity have also been examined. Several key features of the peptide backbone conformation responsible for binding and transduction have been further studied by theoretical calculations and computer modeling (energy minimization) using the Sybyl program.
Structure-activity relationship analysis: Developing glucagon agonists and antagonists for studies of glucagon action in normal and diabetic statesHruby, Victor J.; Azizeh, Bassem Yousef (The University of Arizona., 1996)Several glucagon analogues containing substitutions in the N-terminal region, in particular residues 1, 5, 6, 9 and 10 (histidine, threonine, phenylalanine, aspartic acid and tyrosine, respectively), were synthesized. In addition four β-methylphenylalanine isomers were introduced at position ten to assess the role of these topographical modifications on hormone activity, and to study the effect of constraint and biased conformational preferences of the side chain moieties on biological activity. All the analogues were synthesized by solid-phase methodology, purified to homogeneity by reverse-phase high-performance liquid chromatography, and characterized by electrospray mass spectroscopy, amino acid analysis and thin layer chromatography. Following characterization they were analyzed using rat liver plasma membranes for receptor-binding affinity as well as their ability to stimulate adenylate cyclase. Structure-activity relationship analysis provided critical information about the conformational, chemical and structural properties of amino acid residues required for receptor recognition and signal transduction in the glucagon sequence. His¹ was confirmed to operate along with Asp⁹ for the activation and binding to the glucagon receptor. These new findings should permit the design of more pure and potent glucagon receptor antagonists by focusing on the role of Phe⁶ and other residues in the N-terminal region. A newly developed assay for examining low levels of cAMP accumulation in response to glucagon antagonists, agonists and partial agonists was developed. Previously reported glucagon receptor antagonists had partial agonist activity in rat hepatocytes. This assay system, in conjunction with binding and adenylate cyclase studies in both hepatocytes and liver plasma membranes, redefines the major characteristics of pure glucagon antagonists. The most potent glucagon receptor antagonist [des-His¹, des-Phe⁶, Glu⁹]glucagon-NH₂ was studied using conformational analysis and 2D NMR techniques to analyze the secondary structure of the analogue. Proton resonance assignments using COSY, NOESY and TOCSY in d₆-DMSO were made.
Combinatorial Targeting of the Glucagon-Like Peptide-1 And Sulfonylurea-1 Receptors Using a Complimentary Multivalent Glucagon-Like Peptide-1/Glibenclamide Ligand for the Improvement of β-Cell Targeting Agents and Diabetic TreatmentLynch, Ronald M.; Hart, Nathaniel; Lynch, Ronald M.; Limesand, Sean; Brooks, Heddwen; Konhilas, John; Dokken, Betsy (The University of Arizona., 2013)A scourge of Type I and Type II diabetes impacts the health of hundreds of millions worldwide. The number and prevalence of diabetics are expected to rise dramatically in the next two decades. Diabetes is defined by chronic hyperglycemia which can result in a number of detrimental and costly metabolic, renal, cardiovascular and neurological disorders. Identification of at risk individuals and effective blood glucose management are critical to improving diabetic outcomes and preventing hyperglycemic complications. Diabetes prevention and treatment is limited by the understanding of islet function and mass in the diabetogenic and diabetic state. The islets of Langerhans are dispersed throughout the pancreas and comprise <2% of the pancreatic mass. The reclusive nature of islet cells presents unique challenges understanding disease development. No agent capable of exclusively targeting pancreatic β-cells within the islet has been discovered and the lack of targeting agent specificity impedes efforts to: quantify β-cell mass and develop novel therapeutics. We propose β-cell targeting can be improved by targeting unique combinations of receptors simultaneously with multivalent ligands. A synthetic multivalent agent composed of two β-cell specific diabetic therapeutics, glucagon-like peptide-1 (GLP-1) and glibenclamide (Glb), targeted against the GLP-1R and the sulfonylurea-1 receptor (SUR1) is a lead compound for the development of specific bi-functional islet cell targeting agents for use in the in vivo detection and treatment of β -cells. Herein, we describe the synthesis and initial characterization of a heterobivalent ligand composed of GLP-1 coupled to Glb. The heterobivalent ligand binds to an unaltered β-cell line with increased specificity relative to a human pancreatic exocrine cell line. Additionally, receptor cross-linking modifies β-cell signaling. Exposure of β-cells to the heterobivalent ligand results in antagonism of SUR1-Ca²⁺ signaling and equipotent agonism of GLP-1R-cAMP signaling, in comparison to the cognate monomeric ligands (Glb and GLP-1). Perturbations in intracellular signaling modifies β-cell insulin secretion resulting in decreased basal insulin secretion and with maintained yet reduced ability to potentiate β-cell glucose stimulated insulin secretion. GLP-1/Glb β-cell specificity and functional modulation suggests combinatorial receptor targeting is an effective strategy for the development of bi-functional cell-specific targeting agents, warranting further investigation and optimization.