Be positive! More than a state of mind behind the thermodynamic study of metal complexes with novel calcitermin peptide derivatives

The antimicrobial peptide calcitermin (VAIALKAAHYHTHKE) has been previously studied by our research group to evaluate its metal chelating ability and bioactivity against various pathogenic microorganisms1. To improve the antimicrobial efficacy, some modifications have been introduced in its sequence with the purpose of (i) improving membrane permeabilization – by increasing the number of positive charges —, (ii) improving the affinity for metal ions, and (iii) increasing the proteolytic stability; however, preserving the lipophilic N-terminal portion of the peptide and its original amphiphilic characteristics. Therefore, we studied the formation of Zn(II) and Cu(II) complexes with the calcitermin derivatives A7R and A8R, where the alanine residues in position 7 and 8 are replaced with arginines, which are known to enhance the antibacterial activity.2 The A7H analogue has been also considered, to obtain a chelating sequence with four histidines in alternate position. Indeed, the metal-binding affinity not only increases with the number of histidines but, particularly for zinc ion, it is also higher for histidines separated by a single amino acid rather than separated by several amino acids or consecutive3. In addition, the A7R, A8R and AH7 peptides, protected by acetylation and amidation at the N- and C-terminus, respectively, have been studied to verify the impact of the terminal protection on the peptide properties, including metal coordination and proteolytic stability. After peptide synthesis and purification, a deep investigation of complex formation equilibria and coordination chemistry of the formed species has been obtained by means of potentiometric titrations, mass spectrometry, UV-Vis and circular dichroism. Enzymatic stability assays provided the half-lives in human plasma of the investigated peptides.