Overcoming the challenge of antibiotic resistance requires innovative strategies such as stable, metal-enhanced AMPs [1]. Human salivary antimicrobial peptides are key components of the innate immune defense, exhibiting natural antimicrobial properties against a broad spectrum of pathogens. The antimicrobial activity of these peptides is significantly enhanced upon coordination with metal ions such as copper or zinc, stabilizing their structure and increasing biological efficacy [2]. This metal-dependent activation mechanism is particularly relevant for proline-rich sequences, which often lack a defined secondary structure yet display potent activity through specific molecular interactions. Despite their advantages, the therapeutic application of AMPs is often limited by their susceptibility to enzymatic degradation in a physiological environment. To address this, current research focuses on designing peptidomimetics—structurally modified analogs that retain or even enhance antimicrobial activity while exhibiting improved resistance to proteolysis [3]. In this study, we investigate the enzymatic stability, thermodynamics, coordination behavior, structural properties, and antimicrobial activity of Cu(II) and Zn(II) complexes with salivary proline-rich peptides and their D-amino acid-substituted analogs (Figure). A comprehensive analytical approach was employed to investigate the coordination behavior and stability of metal–peptide and metal–peptidomimetic complexes. This included potentiometric titration, a range of spectroscopic techniques (UV-Vis, circular dichroism, and electron paramagnetic resonance), mass spectrometry, and high-performance liquid chromatography (HPLC). The antimicrobial potential of the studied compounds was evaluated through biological assays. Our findings reveal that metal coordination preferences vary depending on the applied modifications. Enantiomeric substitution of amino acids significantly enhances the thermodynamic stability of Cu(II) and Zn(II) complexes, while the enzymatic stability of partially modified peptides remains unchanged. In contrast, the fully D-amino acid analog exhibits exceptional resistance to proteolysis. Among all analyzed peptides, the LPPSPNNPK peptide demonstrated the strongest antimicrobial activity. It is characterized by its high proline content, which likely contributes to enhanced biological performance through increased rigidity and potential for favorable interactions with microbial membranes. Additionally, only the protachykinin-derived fragment adopts a defined secondary structure, forming a polyproline II (PPII) helix, possibly playing a role in facilitating specific coordination geometry and improved molecular recognition or membrane interaction. Overall, this study highlights the importance of enantiomeric substitutions and proline-rich motifs in tuning the stability and activity of antimicrobial peptides. These strategies provide a rational basis for designing next-generation antimicrobial agents with enhanced therapeutic potential.
Enhancing Antimicrobial Activity of Salivary Proline-rich Peptides via Metal Coordination and D-Amino Acid Substitution
Denise BELLOTTI;
2025
Abstract
Overcoming the challenge of antibiotic resistance requires innovative strategies such as stable, metal-enhanced AMPs [1]. Human salivary antimicrobial peptides are key components of the innate immune defense, exhibiting natural antimicrobial properties against a broad spectrum of pathogens. The antimicrobial activity of these peptides is significantly enhanced upon coordination with metal ions such as copper or zinc, stabilizing their structure and increasing biological efficacy [2]. This metal-dependent activation mechanism is particularly relevant for proline-rich sequences, which often lack a defined secondary structure yet display potent activity through specific molecular interactions. Despite their advantages, the therapeutic application of AMPs is often limited by their susceptibility to enzymatic degradation in a physiological environment. To address this, current research focuses on designing peptidomimetics—structurally modified analogs that retain or even enhance antimicrobial activity while exhibiting improved resistance to proteolysis [3]. In this study, we investigate the enzymatic stability, thermodynamics, coordination behavior, structural properties, and antimicrobial activity of Cu(II) and Zn(II) complexes with salivary proline-rich peptides and their D-amino acid-substituted analogs (Figure). A comprehensive analytical approach was employed to investigate the coordination behavior and stability of metal–peptide and metal–peptidomimetic complexes. This included potentiometric titration, a range of spectroscopic techniques (UV-Vis, circular dichroism, and electron paramagnetic resonance), mass spectrometry, and high-performance liquid chromatography (HPLC). The antimicrobial potential of the studied compounds was evaluated through biological assays. Our findings reveal that metal coordination preferences vary depending on the applied modifications. Enantiomeric substitution of amino acids significantly enhances the thermodynamic stability of Cu(II) and Zn(II) complexes, while the enzymatic stability of partially modified peptides remains unchanged. In contrast, the fully D-amino acid analog exhibits exceptional resistance to proteolysis. Among all analyzed peptides, the LPPSPNNPK peptide demonstrated the strongest antimicrobial activity. It is characterized by its high proline content, which likely contributes to enhanced biological performance through increased rigidity and potential for favorable interactions with microbial membranes. Additionally, only the protachykinin-derived fragment adopts a defined secondary structure, forming a polyproline II (PPII) helix, possibly playing a role in facilitating specific coordination geometry and improved molecular recognition or membrane interaction. Overall, this study highlights the importance of enantiomeric substitutions and proline-rich motifs in tuning the stability and activity of antimicrobial peptides. These strategies provide a rational basis for designing next-generation antimicrobial agents with enhanced therapeutic potential.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
