Stepwise structural optimisation and functional evaluation of dermaseptin-DI2, an antimicrobial peptide identified from <i>Phyllomedusa tomopterna</i>

A stepwise optimisation framework was developed to enhance the therapeutic potential of antimicrobial peptides (AMPs) against multidrug-resistant (MDR) pathogens, integrating C-terminal truncation to improve therapeutic index (TI), targeted amino acid substitutions to enhance serum stability, and hydrocarbon stapling–prodrug integration to boost salt stability while mitigating host toxicity. In Chapter 3, dermaseptin-DI2, a frog-skin-derived peptide, underwent rational two-step C-terminal truncation, yielding DI2(1–19) with a > 50-fold TI improvement over the parent while retaining potent, broad-spectrum activity and rapid bactericidal kinetics.

Both peptides maintained stable MICs over 15 passages, outperforming first-line antibiotics, although DI2(1–19) showed reduced ionic stability and antibiofilm potency. In Chapter 4, DI2(1–19) was further optimised at the proteolytic site Lys⁴ via machine learning-guided substitutions with unnatural Lys analogues. Among these, [Orn⁴]DI2(1–19) and [Dab⁴]DI2(1–19) retained bactericidal speed, inhibited biofilms, and improved activity retention in human serum by up to seven-fold over DI2(1–19).