Heterogeneous-Backbone Proteomimetic Analogues of Lasiocepsin,a Disulfide-Rich Antimicrobial Peptide with a Compact Tertiary Fold

The emergence of resistance to clinically used antibiotics bybacteria presents a significant problem in public health. Natural antimicrobialpeptides (AMPs) are a valuable source of antibiotics that act by a mechanism lessprone to the evolutionary development of resistance. In an effort to realize thepromise of AMPs while overcoming limitations such as poor biostability,researchers have sought sequence-defined oligomers with artificial amide-basedbackbones that show membrane-disrupting functions similar to natural agents.Most of this precedent has focused on short peptidomimetic analogues ofunstructured chains or secondary folds; however, the natural antimicrobial arsenalincludes a number of small- and medium-sized proteins that act via an orderedtertiary structure. Generating proteomimetic analogues of these scaffolds poses a challenge due to the increased complexity of thetarget for mimicry. Here, we report the development of heterogeneous-backbone variants of lasiocepsin, a 27-residue disulfide-richAMP found in bee venom that adopts a compact tertiary fold. Iterative cycles of design, synthesis, and biological evaluation yieldedanalogues of the natural domain with similar to 30 to 40% artificial backbone content, comparable antibacterial activity, reduced host celltoxicity, and improved stability to proteolytic degradation. High-resolution structures determined for several variants by NMRprovide insights into the interplay among backbone composition, tertiary fold, and biological properties. Collectively, the resultsreported here broaden the scope of protein functional mimicry by artificial backbone analogues of tertiary folding patterns andsuggest protein backbone engineering as a means to tune protein function by exerting site-specific control over protein foldedstructure.