Pengyun Li

Design, synthesis and crystallization of a novel glucagon analog as a therapeutic agent

Glucagon and glucagon-like peptide 1 (GLP-1) are drugs or drug candidates for the treatment of metabolic diseases such as diabetes and obesity. The native hormones have pharmacological deficiencies such as short half-life and poor solubility. A novel glucagon receptor agonist named glucagon-Cex has been designed, synthesized and crystallized. This peptide was highly soluble under physiological conditions and crystallized readily. The crystal diffracted X-rays to 2.2 A resolution and the diffraction was consistent with space group P23, with unit-cell parameters a = b = c = 48.20 A, alpha = beta = gamma = 90.0 degrees. The crystals were suitable for a full structural determination to reveal the conformational differences between glucagon-Cex and the native hormone.

Charge inversion at position 68 of the glucagon and glucagon-like peptide-1 receptors supports selectivity in hormone action

Glucagon and glucagon‐like peptide‐1 (GLP‐1)are two structurally related hormones that acutely regulate glucose control in opposite directions through homologous receptors. The molecular basis for selectivity between these two hormones and their receptors is of physiological and medicinal importance. The application of co‐agonists to enhance body weight reduction and correct multiple abnormalities associated with the metabolic syndrome has recently been reported. Substitution of amino acids 16, 18, and 20 in glucagon with those found in GLP‐1 and exendin‐4 were identified as partial contributors to balanced, high potency receptor action. The amidation of the C‐terminus was an additional glucagon‐based structural change observed to be of seminal importance to discriminate recognition by both receptors. In this work, the molecular basis for receptor selectivity associated with differences in C‐terminal peptide sequence has been determined. A single charge inversion in glucagon and GLP‐1 receptor sequence at position 68* was determined to significantly alter hormone action. Changing E68* in GLP‐1R to the corresponding Lys of GCGR reduced receptor activity for natural GLP‐1 hormones by eightfold. The enhanced C‐terminal positive charges in GLP‐1 peptides favor the native receptors negative charge at position 68*, while the unfavorable interaction with the C‐terminal acid of native glucagon is minimized by amidation. The extension of these observations to other glucagon‐related hormones such as oxyntomodulin and exendin, as well as other related receptors such as GIPR, should assist in the assembly of additional hormones with broadened pharmacology. Copyright

Crystallization and Preliminary X-Ray Analysis of Anti-Obesity Peptide Hormone Oxyntomodulin

Oxyntomodulin is a proglucagon-derived gut hormone that reduces food intake and body weight, thus represents a potential therapy for obesity. We synthesized and crystallized oxyntomodulin. The crystal diffracts x-ray to 2.4 A resolution and belongs to space group P2(1)3 with unit-cell parameters a=b=c= 48.44 A, alpha=beta=gamma=90 degrees . Preliminary analysis indicates a trimer packing in one asymmetric unit.

Fibroblast activation protein (FAP) as a novel metabolic target

Objective Fibroblast activation protein (FAP) is a serine protease belonging to a S9B prolyl oligopeptidase subfamily. This enzyme has been implicated in cancer development and recently reported to regulate degradation of FGF21, a potent metabolic hormone. Using a known FAP inhibitor, talabostat (TB), we explored the impact of FAP inhibition on metabolic regulation in mice. Methods To address this question we evaluated the pharmacology of TB in various mouse models including those deficient in FGF21, GLP1 and GIP signaling. We also studied the ability of FAP to process FGF21 in vitro and TB to block FAP enzymatic activity. Results TB administration to diet-induced obese (DIO) animals led to profound decreases in body weight, reduced food consumption and adiposity, increased energy expenditure, improved glucose tolerance and insulin sensitivity, and lowered cholesterol levels. Total and intact plasma FGF21 were observed to be elevated in TB-treated DIO mice but not lean animals where the metabolic impact of TB was significantly attenuated. Furthermore, and in stark contrast to naïve DIO mice, the administration of TB to obese FGF21 knockout animals demonstrated no appreciable effect on body weight or any other measures of metabolism. In support of these results we observed no enzymatic degradation of human FGF21 at either end of the protein when FAP was inhibited in vitro by TB. Conclusions We conclude that pharmacological inhibition of FAP enhances levels of FGF21 in obese mice to provide robust metabolic benefits not observed in lean animals, thus validating this enzyme as a novel drug target for the treatment of obesity and diabetes.

A hydrophobic site on the GLP-1 receptor extracellular domain orients the peptide ligand for signal transduction.

Structure-function studies have analyzed substitutions within the glucagon-like peptide-1 (GLP-1) sequence that increase resistance to proteolysis, however, the investigation into how such substitutions alter interactions at the GLP-1 receptor (GLP-1R) has captured less attention. This work describes our efforts at identifying relevant interactions between peptide ligands and the GLP-1R extracellular domain that contribute to the positioning of the peptide N-terminus for receptor activation. Alanine substitutions at hydrophilic (Glu127⁎ and Glu128⁎) and hydrophobic (Leu32⁎) GLP-1R residues were previously shown to differentially interact with GLP-1 and exendin-4. We examined if these receptor residues influence the activity of GLP-1- and exendin-4-based peptides containing either alanine or glycine at position 2. Additionally, a series of glucagon-based peptides were studied to determine how the central to C-terminal region affects activity. Our results suggest that peptide binding to the GLP-1R is largely driven by hydrophobic interactions with the extracellular domain that orient the N-terminus for activation.

Synthetic Advances in Insulin-like Peptides Enable Novel Bioactivity

Insulin is a miraculous hormone that has served a seminal role in the treatment of insulin-dependent diabetes for nearly a century. Insulin resides within in a superfamily of structurally related peptides that are distinguished by three invariant disulfide bonds that anchor the three-dimensional conformation of the hormone. The additional family members include the insulin-like growth factors (IGF) and the relaxin-related set of peptides that includes the so-called insulin-like peptides. Advances in peptide chemistry and rDNA-based synthesis have enabled the preparation of multiple insulin analogues. The translation of these methods from insulin to related peptides has presented unique challenges that pertain to differing biophysical properties and unique amino acid compositions. This Account presents a historical context for the advances in the chemical synthesis of insulin and the related peptides, with division into two general categories where disulfide bond formation is facilitated by native conformational folding or alternatively orthogonal chemical reactivity. The inherent differences in biophysical properties of insulin-like peptides, and in particular within synthetic intermediates, have constituted a central limitation to achieving high yield synthesis of properly folded peptides. Various synthetic approaches have been advanced in the past decade to successfully address this challenge. The use of chemical ligation and metastable amide bond surrogates are two of the more important synthetic advances in the preparation of high quality synthetic precursors to high potency peptides. The discovery and application of biomimetic connecting peptides simplifies proper disulfide formation and the subsequent traceless removal by chemical methods dramatically simplifies the total synthesis of virtually any two-chain insulin-like peptide. We report the application of these higher synthetic yield methodologies to the preparation of insulin-like peptides in support of exploratory in vivo studies requiring a large quantity of peptide. Tangentially, we demonstrate the use of these methods to study the relative importance of the IGF-1 connecting peptide to its biological activity. We report the translation of these finding in search of an insulin analog that might be comparably enhanced by a suitable connecting peptide for interaction with the insulin receptor, as occurs with IGF-1 and its receptor. The results identify a unique receptor site in the IGF-1 receptor from which this enhancement derives. The selective substitution of this specific IGF-1 receptor sequence into the homologous site in the insulin receptor generated a chimeric receptor that was equally capable of signaling with insulin or IGF-1. This novel receptor proved to enhance the potency of lower affinity insulin ligands when they were supplemented with the IGF-1 connecting peptide that similarly enhanced IGF-1 activity at its receptor. The chimeric insulin receptor demonstrated no further enhancement of potency for native insulin when it was similarly prepared as a single-chain analogue with a native IGF-1 connecting peptide. These results suggest a more highly evolved insulin receptor structure where the requirement for an additional structural element to achieve high potency interaction as demonstrated for IGF-1 is no longer required.

Molecular elements in FGF19 and FGF21 defining KLB/FGFR activity and specificity

Objective To signal, FGF19 and FGF21 require co-receptor βKlotho (KLB) to act in concert with FGF receptors, and yet there is appreciable variance in the C-terminal sequences of these two novel metabolic hormones where binding is believed to be primary. We seek to determine the functional consequences for these amino acid differences and determine whether such information can be used to design high potency antagonists and agonists. Methods We employed a functional in vitro assay to identify C-terminal protein fragments capable of fully blocking KLB-mediated FGF19 and 21 receptor signaling. The key residues in each hormone responsible for support full bioactivity were identified through peptide-based Ala-scanning. Chemical optimization of the peptides was employed to increase their antagonistic potency. An optimized sequence as a substituted part of a full length FGF21 was assessed for enhanced FGFR/KLB-mediated agonism using tissue culture and obese mice. Results C-terminal FGF19 and FGF21 peptides of relatively short length were observed to potently inhibit the activity of these two hormones, in vitro and in vivo. These FGFs of different sequence also demonstrated a striking conservation of structural determinants to maintain KLB binding. A single C-terminal amino acid in FGF19 was observed to modulate relative activity through FGFR1 and FGFR4. The substitution of native FGF21 C-terminal sequence with a peptide optimized for the highest antagonistic activity resulted in significantly enhanced FGF potency, as measured by in vitro signaling and improvements in metabolic outcomes in diet-induced obese mice. Conclusions We report here the ability of short C-terminal peptides to bind KLB and function as antagonists of FGF19 and 21 actions. These proteins maintain high conservation of sequence in those residues central to KLB binding. An FGF21 chimeric protein possessing an optimized C-terminal sequence proved to be a super-agonist in delivery of beneficial metabolic effects in obese mice.