Impact of C- and N-terminal protection on the stability, metal chelation and antimicrobial properties of calcitermin

Abstract

The main limitation to the use of antimicrobial peptides (AMPs) as regular drugs, against antibiotic and antifungal resistance, mainly relates to their rapid degradation by proteolytic enzymes. The introduction of suitable structural changes in the peptide chain can make the peptide less susceptible to the action of proteases, thus overcoming this problem. To improve the plasma stability of calcitermin, a metal-chelating AMP present in the human respiratory tract and investigated in the present study, C- and/or N- terminal modifications have been introduced in the native sequence. Evaluation of peptide stability has been performed to determine the half-life times in human plasma of both native calcitermin and its derivatives. However, the protection of the peptide termini can also affect its metal coordination behaviour. Thus, the characterization of Zn2+ and Cu2+ complexes has been performed by means of several techniques, including potentiometry, high-resolution mass spectrometry, UV–Vis, circular dichroism and EPR. On the basis of the obtained results, it was possible to compare the biological activity of the studied systems, taking into account both the metal-binding ability and the peptide stability to search for a link among them. A significant result of this study is that the N-terminal protection increases the calcitermin half-life over seven times and the formation of metal complexes confers resistance towards degradation almost doubling its half-life.

Metal coordination governs the antimicrobial efficacy of calcitermin derivatives

“…4 and is compared with the proteolytic susceptibility of WT calcitermin (18 min). 13 The degradation profile of the three Ala-to-Ser derivatives follows that of the native peptide and the concentration exponentially decreases with a half-life period ( t 1/2 ) of 19 ± 4 min for A7S/A8S , 21 ± 4 min for A7S and 22 ± 4 min for A8S . According to the experimental data, the Ala-to-Ser substitutions in positions 7 and 8 do not significantly enhance the resistance of calcitermin in human plasma.…”

“…It is a well-studied 15-mer AMP (VAIALKAAHYHTHKE, WT) corresponding to the C-terminal domain of calgranulin C, a pro-inflammatory protein of the S100 family. The presence of three alternating histidine residues (His9, His11 and His13) and the free terminal amino and carboxyl groups enables the coordination of metal ions like Zn 2+ and Cu 2+ , 12 which also enhances its antimicrobial activity against Candida albicans , 13 and common bacteria like Staphylococcus aureus and Enterococcus faecalis at acidic pH, a typical condition in the case of inflammation. 14 It also has the potential to adopt a helical conformation in membranes.…”

“…14 It also has the potential to adopt a helical conformation in membranes. 13 The lowering of the pH value contributes to the protonation of the three histidine residues, thus favouring the interaction of calcitermin with the negatively charged groups present on the surface of bacterial cells. 12,14,15…”

Capping motifs in antimicrobial peptides and their relevance for improved biological activities

“…With all this evidence, Hst-5 cannot be classified as belonging to class II; instead, it appears to be able to act in class I or III. Calcitermin (Val-Ala-Ile-Ala-Leu-Lys-Ala-Ala-His-Tyr-His-Thr-His-Lys-Glu) corresponds to the last 15 residues at the C-terminus of calgranulin C, a member of the S100 family of antibacterial proteins produced by neutrophils, monocytes, and keratinocytes (Gottsch et al, 1999;D'Accolti et al, 2023). This AMP shows activity against several pathogens (E. coli, P. aeruginosa, S. aureus, S. epidermidis, E. faecalis, C. albicans, and L. monocytogenes), depending on the conditions (Cole et al, 2001).…”

Design, Synthesis and Antimicrobial Potential of Conjugated Metallopeptides Targeting DNA

“…The conformation of the peptide has a substantial impact on the coordination equilibria in the metal-peptide system, from both a thermodynamic and a structural perspective [10]. Aliphatic or aromatic chains form a spatial shield that prevents the hydrolysis of N-metal bonds and increases the structural strength of the complex molecule [11].…”