The effect of Ala-to-Ser substitution on calcitermin derivatives

Nowadays the phenomenon of antimicrobial resistance (AMR) is one of the most urgent threats to public health, causing serious problems in the prevention and successful treatment of many diseases. AMR occurs when microorganisms including bacteria, viruses, fungi and parasites become able to adapt and grow in the presence of previously effective medications. Despite different actions taken in recent decades to tackle this problem, the trend of global AMR demonstrates no improvements. Misusing and overusing antibacterial agents are considered the major reasons behind the drug-resistance emergence, together with spontaneous evolution and mutation of pathogenic microorganisms. In order to overcome the global AMR crisis, the use of antimicrobial peptides (AMPs) represent a promising strategy for the design of new drugs. AMPs are phylogenetically ancient biomolecules with a broad spectrum of activity and scarce attitude to induce antimicrobial resistance. They are known to be effective against a wide variety of pathogens, like Gram-positive and Gram-negative bacteria, fungi, viruses and even cancer cells, and are present in all living organisms. Their activity can be expressed in different ways, including the interaction with cell membranes and through the innate immune response termed “nutritional immunity”. Thanks to this mechanism, AMPs sequestrate essential metal micronutrients such as Zn(II), Cu(II), Mn(II), Fe(II) or Ca(II), which are fundamental for pathogen subsistence. Among several AMPs, we are interested in calcitermin [1, 2], a human 15 amino-acid antimicrobial peptide (VAIALKAAHYHTHKE) corresponding to the C-terminal domain of calgranulin C, a pro-inflammatory protein of the S100 family. Calcitermin presents an effective metal-binding domain with three alternated histidine residues (His 9, His11 and His13) and the free terminal amino and carboxyl groups. It exhibits an increased microbicide activity when Zn(II) or Cu(II) ions are present in the culture medium. In order to improve the metal binding ability and the biologic activity of calcitermin, we synthesized and studied the analogues VAIALKSAHYHTHKE (A7S), VAIALKASHYHTHKE (A8S) and VAIALKSSHYHTHKE (A7S/A8S), in which the alanine residues in position 7 and/or 8 have been replaced with a serine. In fact, several studies carried out on metal chelating peptides have previously shown that the presence of one or more serine residues in the proximity of coordinated histidines stabilized the Cu2+ complexes when the metal ion begins to interact with the amides of the peptide chain [3]. We also studied the complexes with zinc ion, which is the most important endogenous metal ion capable to interact with calcitermin. The characterization of the complexes has been achieved by means of mass spectrometry, potentiometry, UV-Vis spectrophotometry, circular dichroism, electron paramagnetic resonance. The results show that all the investigated peptides are efficient ligands for the considered metal ions and can form stable mono-nuclear complexes. The proteolytic stability of these three peptides has been tested in human plasma by means of HPLC, evaluating the degradation content after different times of incubation at 37 °C. The results show that they are slightly more stable than native calcitermin. Finally, the antimicrobial activity of calcitermin derivatives and their metal complexes with Zn(II) and Cu(II), has been studied in vitro against human pathogenic strains. Financial support of the Polish National Science Centre (UMO-2020/37/N/ST4/03165), of the University of Ferrara (FAR 2021) and of the COST Action CA18202-Nectar is gratefully acknowledge.