How terminal protection affects the chemical and biological properties of the antimicrobial calcitermin

The discovery of antibiotics changed modern medicine, both drastically reducing the severity of many bacterial diseases, and allowing spectacular progress in other fields, such as chemotherapy, surgery, and organ transplant. However, the abuse and misuse of these excellent drugs has led to an increase of antimicrobial resistance (AMR), a phenomenon which occurs when microorganisms learn to adapt and grow in the presence of previously effective medications. AMR would require the continuous discover and introduction of new antibiotics, but, unfortunately, in the last decades, the investments in this field have been highly insufficient to face the clinical and financial burden due to AMR [1]. Antimicrobial peptides (AMPs) represent a promising base for developing new drugs since they present a broad spectrum of activity and a scarce attitude to induce antimicrobial resistance. In fact, their activity can be exerted through different mechanisms of action such as the interaction with cell membranes and the innate immune response termed “nutritional immunity”. AMPs act as mediators of the immune defence through the sequestration of metallic nutrients such as Zn2+, Cu2+, Mn2+ or Ca2+, interfering with the homeostasis of the pathogenic cells [2]. On the other hand, AMPs present the drawback of a metabolic instability since they are subject to degradation by both human and pathogenic proteolytic enzymes. Calcitermin is 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 [3]. It presents an effective metal-binding domain with three alternated histidine residues (His9, His11 and His13) and the free terminal amino and carboxyl groups. Several studies showed an increased microbicide activity when Zn2+ and Cu2+ ions are present in the culture medium. Our research group have recently determined the stability of this calcitermin in human plasma which corresponds to a half-life of 18 minutes [4]. In order to extend this lifetime, i.e. to obtain a better proteolytic resistance, without losing the antimicrobial properties, several strategies can be employed. To increase the resistance towards exopeptidases (enzyme that catalyse the cleavage of the terminal peptide bonds), the chemical modification of one or both the peptide ends can be applied. To this aim, we protected the amino- and carboxyl-termini of calcitermin by acetylation and amidation respectively [4], obtaining the following derivatives: Ac-VAIALKAAHYHTHKE, Ac-VAIALKAAHYHTHKE-NH2 and VAIALKAAHYHTHKE-NH2. Of note, the introduction of such terminal protection preserves the metal binding site of calcitermin, corresponding to the histidine motif –HxHxH-. We investigated the complex-formation equilibria of these mutants with Zn2+ and Cu2+ by means of different experimental techniques: mass spectrometry, potentiometry, UV-Vis spectrophotometry, circular dichroism and electron paramagnetic resonance. In addition, the peptide stability in human plasma was determined by HPLC and the antimicrobial activity towards different potential pathogens has also been investigated. The results show that with both copper and zinc, the most stable complexes are always those formed by the native calcitermin, throughout the explored pH range, suggesting an evident contribution of the amino and carboxyl termini. As expected, the acetylation of the Nterminal confers a much longer half-life with respect to peptides with free amino terminus. Interestingly, the formation of both copper and zinc complexes almost doubled the half-life of calcitermin in human plasma. Finally, encouraging results have been obtained for the Zn2+ complexes of the protected peptides against Candida albicans.