Abstract:
Peptides offer several advantages in drug discovery due to their ability to mimic natural biological molecules, enabling high target specificity and potency. Their biological activity is influenced by sequence, structure, and length. However, a major drawback of peptide-based therapeutics is their poor proteolytic stability. In this study, we investigated whether proteolytic stability can be improved while retaining biological activity by incorporating opposite-chirality D-amino acids and non-ribosomal g-amino acids. We compared L-α-peptides and their D-α-peptide analogues for antimicrobial activity. The 15-residue peptide L-α-[KLAKLAKKLAKLAKL] and its D-analogue exhibited no significant antibacterial activity. However, increasing the peptide length to 18- and 21-mers resulted in potent antibacterial activity. Circular dichroism (CD) analysis confirmed that both peptide types adopted helical conformations in the presence of SDS. Both peptides also induced liposomal leakage at very low concentrations. Interestingly, cytotoxicity studies revealed that while the L-series showed low to moderate cytotoxicity, the D-series displayed very high cytotoxicity, comparable to the positive control vancomycin. To extend this investigation, we examined whether longer KLA peptides and their ααg-hybrid analogues could inhibit Aβ42 aggregation in vitro. Both α-peptides and ααg-hybrids effectively inhibited aggregation. Notably, ααg-hybrid peptides exhibited lower cytotoxicity compared to their α-peptide counterparts. Encouraged by this, we designed a series of ααg-hybrid peptides containing g-valine (g-Val) and g-leucine (g-Leu) residues, along with their all-α analogues. The 15-residue peptides KLV, KLgV, KLL, and KLgL demonstrated excellent antibacterial activity against ESKAPE pathogens. There no much improvement in the activity with increasing length of the peptides. Importantly, ααγ-hybrid peptides showed low cytotoxicity, minimal hemolytic activity, and a favorable therapeutic index. They also exhibited enhanced stability against proteases such as trypsin and chymotrypsin, as well as extended serum stability. Mechanistic studies revealed that these peptides disrupt bacterial membranes and also bind to bacterial DNA. Further investigations indicated that these peptides can function as ion channels at very low concentrations. In summary, compared to conventional α-peptides, ααγ-hybrid peptides demonstrate superior antibacterial activity, significantly lower cytotoxicity, and enhanced stability. These hybrid peptides represent promising candidates for the treatment of multidrug-resistant bacterial infections.
