Understanding the effect of amino- and carboxyl-terminal protection on the stability and metal chelation properties of the antimicrobial peptide calcitermin

Antimicrobial peptides (AMPs) are promising candidates for the design of new therapeutic agents against drug-resistant infections and, in this context, calcitermin (VAIALKAAHYHTHKE), a 15-residue AMP present in the human respiratory tract, is of particular interest [1]. Calcitermin has been previously studied to evaluate its chelating ability towards Zn2+ and Cu2+ ions and to investigate its effectiveness against different pathogenic microorganisms [1,2]. This peptide contains a metal-binding domain with three alternated histidine residues (His9, His11 and His13) and the free terminal amino and carboxyl groups. It has been demonstrated that the presence of micromolar concentration of Zn2+ or Cu2+ enhances the microbicidal activity of calcitermin under acidic conditions (pH = 5.4) [2]. Although AMPs are promising antimicrobial agents, the main limitation to their use as regular drugs relates to the rapid degradation by proteolytic enzymes. Introducing structural changes in the peptide chain could be a possible solution to the problem, making the peptide less susceptible to the action of proteases. Therefore, to improve the plasma stability of calcitermin, C- and/or N- terminal modifications have been introduced in the peptide native sequence [3]. The protection of the peptide termini can affect the biological properties and the metal coordination behaviour of wild-type calcitermin, thus requiring further investigations. The first task of this work consists of the characterization of the formed Zn2+ and Cu2+ complexes with the following peptides: Ac-VAIALKAAHYHTHKE, VAIALKAAHYHTHKE-NH2 and Ac-VAIALKAAHYHTHKE-NH2. A deep investigation of the complex formation equilibria and coordination chemistry of the formed species has been obtained by means of several techniques, including potentiometry, high-resolution mass spectrometry, UV-Vis, circular dichroism and EPR. Furthermore, enzymatic stability assays provided the half-life times in human plasma of native calcitermin and its peptide derivatives. Finally, on the basis of the obtained results, it was possible to compare the studied systems, confirming that the N- and C-terminal groups influence not only the plasma stability but also the metal chelating ability of calcitermin.