Decoding the human serum interactome of snake-derived antimicrobial peptide Ctn[15-34]: Toward an explanation for unusually long half-life

Abstract

The inherent propensity to enzymatic degradation of most peptides remains a bottleneck in their therapeutic development. Efficient, early screening methods are necessary for in vitro characterization of the molecular events occurring when peptides get in contact with biological fluids such us plasma. Herein we present an affinity purification/MS approach for mapping peptide serum interactors. We have applied this methodology to identify the serum partners of antibiotic peptide Ctn [15-34], aiming to ascertain the molecular interactions underlying its unusually long half-life (~ 12 h) in human serum. From 42 proteins captured in pull-downs with biotinylated Ctn [15-34] as bait, five are of special interest for their transport/binding properties hence alleged peptide arresting potential. The subset contains two members of the albumin superfamily, two apolipoproteins and a globulin. All five share a binding ability for hydrophobic species, and also bind Ctn [15-34], presumably via its C-terminal hydrophobic section, with affinities in the μM range as shown by surface plasmon resonance. Additionally, our functional enrichment reveals several significant immune-related processes suggesting an immunomodulatory role of Ctn [15-34]. Taken together, this study exemplifies how pharmacoproteomics can be used to analyze bioavailability issues and shed light on the serum interactors ultimately conferring protection to Ctn [15-34] against proteolytic events. SIGNIFICANCE: The affinity purification/MS identification methodology reported here can be viewed as a routine pharmacoproteomic approach to investigate the serum interactome of peptide drugs, identifying proteins affecting bioavailability and thus assisting the peptide drug development process. The specific results described here enlighten the serum stability issues of peptide Ctn [15-34] and ratify its promising future as an anti-infective lead.

Keywords: Affinity purification; Peptide proteolytic resistance; Peptide serum stability; Peptide-protein interaction; Proteomics; Serum carrier protein; Surface plasmon resonance.