Thanks to the broad spectrum of activity and scarce attitude to induce antimicrobial resistance, antimicrobial peptides (AMPs) represent a rational chance to overcome the current drug-resistance crisis and design innovative therapies. One of the major problems related to the AMP-based drugs is their poor proteolytic stability. A commonly accepted strategy to overcome such drawback is to optimize the peptide structure, introducing appropriate modifications that may improve the resistance to degradation. Peptidomimetics can be designed using unnatural occurring building blocks, which can alter the native peptide physicochemical properties without impairing the antimicrobial activity. Among the variety of existing antimicrobial peptides, we are mostly interested in the investigation of metal–related AMPs: several studies have shown that divalent metal ions can modulate the efficacy of some AMPs. A rational and unequivocal explanation of how the metal exactly participate in the expression of the antimicrobial activity is not yet achieved, but there are two suggestions: the AMPs metal sequestration starves the pathogen and prevents its growth; or the metal binding induces changes in the peptide structure modifying its properties. Based on our preliminary studies, among several AMPs known to have better antimicrobial activity when interacting with a metal ion, we identify a possible candidate that will serve as a starting point: calcitermin, an antimicrobial peptide from the human airways (VAIALKAAHYHTHKE), corresponding to the C-terminal domain of calgranulin C. It contains a metal-binding domain with three alternated histidine residues (His9, His11 and His13) and the free terminal amino and carboxyl groups. It exhibits an improved microbicidal activity when Zn(II) or Cu(II) ions are present in the culture medium. In addition, it has the potential to adopt a helical conformation in membranes. Interestingly, calcitermin His-to-Ala mutants – where each histidine residue is replaced with one alanine – have different metal coordination modes, resulting in significant changes in the antimicrobial properties. Promising effects against Gram-positive bacteria are detected, in particular by the H13A complexes; while the substitution of His11 and His13 with alanine reduces the antifungal activity, instead observed for H9A systems. These preliminary results prompted us to focus on calcitermin derivatives where the peptide structure is modified in order to confer higher proteolytic stability. The consequent effects on their antimicrobial activity will be evaluated, both in the presence and absence of metal ions. Changes in the peptide backbone can affect the calcitermin metal-binding behaviour and therefore further investigations on the metal interaction with the most promising synthesized peptidomimetics will be performed, in order to connect their antimicrobial activity with their complex-formation abilities. After a careful evaluation of the enzymatic stability of calcitermin, in order to determine the “weak points” of the chain, the first task of this project will consist of a series of modifications to obtain calcitermin derivatives resistant to proteases. These will include: (i) a peptide sequence containing D-amino acids; the introduction of (ii) β-amino acids, that may modulate the conformational constrain of the system inducing a helical conformation and ultimately affect the biological activity; the addition of (iii) unnatural amino acids, not recognised by peptidases and, eventually, the use of (iv) peptoids. The enzymatic stability must be checked for each mutant together with its antimicrobial properties against bacterial, fungal and viral pathogens, including SARS-CoV-2, in order to select the most promising peptide derivatives. The in vitro stability will be tested on human plasma. The characterization of metal complexes with calcitermin derivatives will be performed by means of several techniques. The stoichiometry of the formed species will be determined through potentiometry and confirmed by high-resolution mass spectrometry. Potentiometry also provides the stability constants of the formed metal complexes. The identification of binding sites and coordination geometries will be achieved by several spectroscopic techniques (NMR, UV-Vis, fluorimetry, CD and EPR). The obtained results will allow us to propose and design new therapeutic antimicrobial strategies based on calcitermin derivatives metal complexes.
A peptidomimetic strategy to improve the antimicrobial properties of calcitermin
Denise Bellotti
Primo
Funding Acquisition
;
2021
Abstract
Thanks to the broad spectrum of activity and scarce attitude to induce antimicrobial resistance, antimicrobial peptides (AMPs) represent a rational chance to overcome the current drug-resistance crisis and design innovative therapies. One of the major problems related to the AMP-based drugs is their poor proteolytic stability. A commonly accepted strategy to overcome such drawback is to optimize the peptide structure, introducing appropriate modifications that may improve the resistance to degradation. Peptidomimetics can be designed using unnatural occurring building blocks, which can alter the native peptide physicochemical properties without impairing the antimicrobial activity. Among the variety of existing antimicrobial peptides, we are mostly interested in the investigation of metal–related AMPs: several studies have shown that divalent metal ions can modulate the efficacy of some AMPs. A rational and unequivocal explanation of how the metal exactly participate in the expression of the antimicrobial activity is not yet achieved, but there are two suggestions: the AMPs metal sequestration starves the pathogen and prevents its growth; or the metal binding induces changes in the peptide structure modifying its properties. Based on our preliminary studies, among several AMPs known to have better antimicrobial activity when interacting with a metal ion, we identify a possible candidate that will serve as a starting point: calcitermin, an antimicrobial peptide from the human airways (VAIALKAAHYHTHKE), corresponding to the C-terminal domain of calgranulin C. It contains a metal-binding domain with three alternated histidine residues (His9, His11 and His13) and the free terminal amino and carboxyl groups. It exhibits an improved microbicidal activity when Zn(II) or Cu(II) ions are present in the culture medium. In addition, it has the potential to adopt a helical conformation in membranes. Interestingly, calcitermin His-to-Ala mutants – where each histidine residue is replaced with one alanine – have different metal coordination modes, resulting in significant changes in the antimicrobial properties. Promising effects against Gram-positive bacteria are detected, in particular by the H13A complexes; while the substitution of His11 and His13 with alanine reduces the antifungal activity, instead observed for H9A systems. These preliminary results prompted us to focus on calcitermin derivatives where the peptide structure is modified in order to confer higher proteolytic stability. The consequent effects on their antimicrobial activity will be evaluated, both in the presence and absence of metal ions. Changes in the peptide backbone can affect the calcitermin metal-binding behaviour and therefore further investigations on the metal interaction with the most promising synthesized peptidomimetics will be performed, in order to connect their antimicrobial activity with their complex-formation abilities. After a careful evaluation of the enzymatic stability of calcitermin, in order to determine the “weak points” of the chain, the first task of this project will consist of a series of modifications to obtain calcitermin derivatives resistant to proteases. These will include: (i) a peptide sequence containing D-amino acids; the introduction of (ii) β-amino acids, that may modulate the conformational constrain of the system inducing a helical conformation and ultimately affect the biological activity; the addition of (iii) unnatural amino acids, not recognised by peptidases and, eventually, the use of (iv) peptoids. The enzymatic stability must be checked for each mutant together with its antimicrobial properties against bacterial, fungal and viral pathogens, including SARS-CoV-2, in order to select the most promising peptide derivatives. The in vitro stability will be tested on human plasma. The characterization of metal complexes with calcitermin derivatives will be performed by means of several techniques. The stoichiometry of the formed species will be determined through potentiometry and confirmed by high-resolution mass spectrometry. Potentiometry also provides the stability constants of the formed metal complexes. The identification of binding sites and coordination geometries will be achieved by several spectroscopic techniques (NMR, UV-Vis, fluorimetry, CD and EPR). The obtained results will allow us to propose and design new therapeutic antimicrobial strategies based on calcitermin derivatives metal complexes.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
