Exploring CXCL10-Derived Antimicrobial Peptides (AMPs): The Role of Polymer Conjugation and Cysteine Oxidation on Properties and Performance

The rise of drug-resistant bacteria presents a serious global health threat, driving the need for novel antimicrobial agents. Two antimicrobial peptides (AMPs) derived from the human chemokine protein CXCL10: stapled-P9, a 22-amino-acid sequence from the C-terminus with an alpha-helical structure, and D8, an unstructured 8-amino-acid sequence, from the N-terminus. To improve the effectiveness of stapled-P9, we developed various peptide-polymer conjugates aiming to reduce toxicity to mammalian cells, increase resistance to enzymatic degradation, and enhance antimicrobial activity. Three different conjugate architectures—linear, star, and comb—were compared using alamarBlue assays to assess their bactericidal activity. Our results indicate that the comb-like conjugates exhibited higher bactericidal activity compared to their linear and star-shaped counterparts, all while maintaining low levels of toxicity to human red blood cells. Peptide D8 (_D-RTVRCTCI), despite lacking a defined structure (unusual for an AMP) also showed broad-spectrum bactericidal activity. To better understand which amino acids influence its function, we exchanged each with _D-alanine, noting decreased activity upon replacing arginine (R), valine (V), isoleucine (I), and cysteine (C). While the reduction in activity from replacing hydrophobic residues (V and I) and cationic residues (R) was expected, given their roles in promoting interaction with hydrophobic and anionic bacterial membranes, we are interested in exploring the role the two cysteines have in D8’s activity. To investigate this, we synthesized three D8-derived peptides with protective caps on one or both cysteines confirming these modifications via MALDI and NMR analysis. Since capping either or both cysteines reduced antimicrobial activity, we examined the relative reactivity of each cysteine to determine if differences in oxidation rates influence D8’s structure and solution behavior. We hypothesized that the cysteine next to the arginine would be more reactive due to the arginine’s positive charge stabilizing the negative charge of the thiolate intermediate. Using Ellman’s reagent, we monitored free (unoxidized) thiol concentrations over time and compared the behavior of D8 with its 5^th cysteine capped versus 7^th cysteine capped. Understanding the role of cysteines and their oxidation in the solution behavior and antimicrobial performance will advance the molecular design of cysteine-containing peptides as novel antimicrobial agents.