Esculentin-2CHa(1–30) and its analogues: stability and mechanisms of insulinotropic action

Abstract

The insulin-releasing effects, cellular mechanisms of action and anti-hyperglycaemic activity of 10 analogues of esculentin-2CHa lacking the cyclic C-terminal domain (CKISKQC) were evaluated. Analogues of the truncated peptide, esculentin-2CHa(1-30), were designed for plasma enzyme resistance and increased biological activity. Effects of those analogues on insulin release, cell membrane integrity, membrane potential, intracellular Ca and cAMP levels were determined using clonal BRIN-BD11 cells. Their acute effects on glucose tolerance were investigated using NIH Swiss mice. d-Amino acid substitutions at positions 7(Arg), 15(Lys) and 23(Lys) and fatty acid (l-octanoate) attachment to Lys at position 15 of esculentin-2CHa(1-30) conveyed resistance to plasma enzyme degradation whilst preserving insulin-releasing activity. Analogues, [d-Arg,d-Lys,d-Lys]-esculentin-2CHa(1-30) and Lys-octanoate-esculentin-2CHa(1-30), exhibiting most promising profiles and with confirmed effects on both human insulin-secreting cells and primary mouse islets were selected for further analysis. Using chemical inhibition of adenylate cyclase, protein kinase C or phospholipase C pathways, involvement of PLC/PKC-mediated insulin secretion was confirmed similar to that of CCK-8. Diazoxide, verapamil and Ca omission inhibited insulin secretion induced by the esculentin-2CHa(1-30) analogues suggesting an action on K and Ca channels also. Consistent with this, the analogues depolarised the plasma membrane and increased intracellular Ca Evaluation with fluorescent-labelled esculentin-2CHa(1-30) indicated membrane action, with internalisation; however, patch-clamp experiments suggested that depolarisation was not due to the direct inhibition of K channels. Acute administration of either analogue to NIH Swiss mice improved glucose tolerance and enhanced insulin release similar to that observed with GLP-1. These data suggest that multi-acting analogues of esculentin-2CHa(1-30) may prove useful for glycaemic control in obesity-diabetes.

Enhanced therapeutic index of an antimicrobial peptide in mice by increasing safety and activity against multidrug-resistant bacteria

“…There have been previous attempts to reduce the lability of AMPs. However, these studies are rare and do not typically result in proof of concept using in vivo models (16,32,33). Our approach includes a rational framework for l-to-d enantiomerization using the cutting sites of a common enzyme (protease) relevant to the intended clinical application (e.g., respiratory infection treatment).…”

The Potential of Frog Skin-Derived Peptides for Development into Therapeutically-Valuable Immunomodulatory Agents

“…Several strategies have evolved to increase the stability of peptide-based agents by increasing their resistance to degradation by peptidases. These include incorporation of D-amino acids or unnatural amino acids such as α-aminoisobutyric acid into the molecule and coupling of the peptide to a fatty acid such as palmitate or octanoate or to polyethylene glycol (reviewed in [ 73 ]). Furthermore, the mechanisms by which frog skin peptides exert their immunomodulatory actions are still obscure.…”

Immunomodulatory, insulinotropic, and cytotoxic activities of phylloseptins and plasticin‐TR from the Trinidanian leaf frogPhyllomedusa trinitatis

“…Preliminary data indicate that plasticin‐TR and phylloseptin‐3.3TR are rapidly degraded in mouse plasma (unpublished observations). Esculentin‐2CHa derivatives containing unnatural amino acids [R7r,K15k,K23k]esculentin‐2CHa (1‐30) and incorporating fatty acids moieties [K 15 ‐octanoate]esculentin‐2CHa (1‐30) are both more potent than the underivatized peptide in vitro and more effective in vivo in terms of their glucose‐lowering and insulin‐releasing activities . Future studies will focus on the development of long‐acting derivatives of plasticin‐TR and phylloseptin‐3.3TR with regard to their therapeutic potential as antiinflammatory and antidiabetic agents.…”