Design, synthesis and biological evaluation of proline-arginine rich hybrid peptides against ESKAPE pathogens by inhibiting bacterial protein synthesis and DNA replication.

The rise of multidrug-resistant (MDR) bacteria, particularly ESKAPE pathogens, has led to the widespread failure of traditional antibiotics, highlighting the urgent need for novel therapeutics. Proline-arginine rich antimicrobial peptides (PrAMPs) have emerged as promising candidates due to their broad-spectrum activity, high cell selectivity, and a lower frequency of resistance development compared to conventional drugs. A strategic target for new antibacterial agents is the bacterial sliding clamp (DnaN), a central component of the DNA replisome. In this study, we designed a series of novel hybrid peptides inspired by a known PrAMP sequences, covalently linked to a peptide inhibitor of DnaN. These hybrids were synthesized via solid-phase peptide synthesis and evaluated against a panel of Gram-positive and Gram-negative bacteria, including MDR strains. Within this library, NHP-13 was identified as the lead analogue, demonstrating potent antimicrobial activity with Minimum Inhibitory Concentrations (MICs) ranging from 15.6 to 125 μM. NHP-13 utilizes a -RRR- (tri-arginine) linker to combine ribosomal-inhibiting and nucleic acid inhibiting sequences. Mode-of-action studies confirmed that NHP-13 functions through intracellular accumulation and inhibition of protein synthesis. Crucially, the peptide showed a non-hemolytic and non-cytotoxic characteristics against mammalian cell lines. Furthermore, the in vivo efficacy was validated in a murine infection model, where NHP-13 showed bacterial clearance of nearly 2 log colony-forming units (CFU) in the tested organs. These results underscore the potential of intracellular hybrid AMPs as safe, effective, and resilient antimicrobial agents for clinical applications.