Stability, toxicity and biological activity of retro, inversed and retro‐inversed glucagon isomers

Abstract

Peptide modifications that improve pharmacological properties are of considerable therapeutic importance. Here we consider the retro (R), inversed (D) and retro-inversed (RI) isomers of glucagon with respect to structure, stability, toxicity and biological activity. Biologically, RI-glucagon demonstrated comparable in vivo activity as L-glucagon with respect to magnitude and duration of blood sugar elevation following i.p. administration to mice. Structurally, the isomers were investigated through circular dichroism (CD) and nanopore analysis. CD demonstrated a conserved potential for formation of secondary structure, which was independent of the direction of the peptide (L vs R; D vs RI) as well as formation of symmetry-related structures for the chiral isomers (L vs D; R vs RI). CD, therefore, discriminated chiral but not directional isomers. Nanopore analysis, which depends on interaction of the peptides with chiral pores, discriminated all four isomers on the basis of unique signatures of bumping and translocation. Nanopore analysis offered greater opportunity than CD to discriminate the isomers although neither technique provided a definitive biomarker of biological activity. Functionally, the R and RI isomers resist proteolytic degradation and none of the isomers possess hemolytic activity or cellular toxicity. Collectively, this investigation highlights the potentials and limitations of CD and nanopore analysis for investigation of peptide isomers as well as offering insight into the structural criteria to mimic peptide biological activity. For this example, retro-inversion, through undefined contributions of increased stability and maintained biological activity, was best suited to mimic the biological activity of the parent peptide.

Superposition of an AC field improves the discrimination between peptides in nanopore analysis

“…8,9 However, for RI-A10, the negative charge is at the C-terminus which will enter the pore first so that the Fmoc is now to the rear of the peptide. 35 As well, at this DC voltage of 100 mV the AC field has only a small effect on the A10 peptide whereas a DC voltage of 60 mV, previously reported, 33 caused a large increase in the number of bumping events. In contrast for RI-A10 the number of translocation events is clearly reduced.…”

“…8,9 RI-A10 has all D-amino acids. 35 The lengths and dipole moments were calculated with Spartan software Current traces such as those of Fig. 2 were converted into blockade current histograms which allows a more detailed analysis of the event profiles.…”

“…3 and 4). 33,35 An equimolar mixture of the two peptides were analyzed without an AC field which gives rise to a broad translocation peak at −59 pA and a minor bumping peak (Fig. 9a).…”

Cu(II) and dopamine bind to α‐synuclein and cause large conformational changes

“…Previous work has shown that nanopore analysis of proteins can detect small conformational changes, because the blockade current is very sensitive to the precise way in which a molecule interacts with the pore . Although the experiments are performed at low concentrations (1 μ m ), which inhibits protein aggregation, even weak binding can be detected.…”