12-Hydroxylauric Acid-Tethered Heterochiral Diphenylalanines: A Promising Antimicrobial Peptide Scaffold for In Vivo...

Microbial infections are among the most critical global issues, imposing a significant financial burden on healthcare systems worldwide. Despite extensive efforts, the development of effective therapeutics remains in its infancy owing to the inability of the available drugs to address the complexity through a unified strategy. On a quest to discover a multifunctional scaffold capable of displaying both self-assembly and antimicrobial activity conjointly, in this study, we represent a rational combinatorial approach designing Compound I (homochiral) and Compound II (heterochiral) that leverage chiral orchestration in diphenylalanine fragments, anchored to an amphiphilic unit - 12-hydroxylauric acid at the N-terminus. Our systematic analysis involving minimum inhibitory concentration (MIC) experiments against B. subtilis (Gram-positive strain) and E. coli (Gram-negative strain) indicated that from a set of two, Compound II exhibited nearly 10 times superior antimicrobial activity. Thus, it was retrieved from the design and examined comprehensively in vitro with the assistance of colony-counting investigation and scanning electron microscopy. Notably, Compound II also demonstrated mechanoresponsive hydrogelation at physiological pH and adopted a β-sheet architecture stabilized by noncovalent interactions as confirmed by temperature-dependent NMR, IR, PXRD, and CD analysis as well as a thioflavin T assay. Moreover, this conformation imparted optimal mechanical strength and proteolytic stability up to 72 h as well as a safety profile, as authenticated by biocompatibility assays on three different cell lines of diversified nature. Finally, the in vivo wound healing experiments showed that Compound II significantly accelerated wound closure by preventing infection of both the microorganisms B. subtilis and E. coli and promoting tissue regeneration with proper hair growth and no scar marks within 7 days of treatment. These findings underscore the potential of Compound II as a promising antimicrobial candidate with a multifaceted mode of action for addressing challenges in microbial infection management.