Multimodal strategies in antimicrobial peptoids discovery: mechanistic insights, structural diversity, and metabolic stability control

Abstract

The escalating crisis of multidrug-resistant (MDR) bacteria provokes the discovery of new antibiotics with novel, resistance-breaking mechanisms of action. Antimicrobial peptoids, synthetic mimics of antimicrobial peptides (AMPs), offer a promising platform due to their enhanced proteolytic stability and versatility in side chain variations that modulate the physicochemical properties. This doctoral thesis presents a multimodal strategy encompassing advanced mechanistic analysis, novel structural engineering, and targeted metabolic control to develop next generation antimicrobial peptoids. In Chapter II, we utilized optical diffraction tomography (ODT), a label-free, real-time 3D imaging technique, to precisely elucidate the antimicrobial mechanisms of action. After establishing a quantitative method for morphological analysis using benchmark AMPs (melittin and buforin-II), we screened an indole- containng peptoids library to identify the potent and selective compound, peptoid 29. ODT analysis, corroborated by fluorescence and eletron microscopy, unequivocally demonstrated that 29 empolys a powerful multi-target mechanism, initiating both membrane disruption and subsequent intracellular biomass flocculation (of proteins and nucleic acids), confirming the value of ODT for identifying multi-mechanistic agents. Chapter III explores metabolic stability control through fluorination, addressing a key pharmacokinetic challenge. We synthesized a series of fluorine-conatining peptoids, investigating the role of fluorine as a hydrogen bioisostere. While the chiral fluorinated monomers in peptoids retained the helical conformation and intrinsic biological activities (antibacterial and hemolytic activity) of the parent peptoids, metabolic stability showed a discernible decrease as the number of fluorine atoms increased. Using the liver S9 fraction and LC-QTOF MS/MS analysis with simplified trimer peptoids, we verified the degradation pathway, thereby confirming that the introduction of fluorine produces a controllable strategy to modulate the metabolic stability of antimicrobial peptoids via specific enzymatic degradation pathways. Chapter IV describes the investigation of dimerization of antimicrobial peptoids and their biological activities with structural characterization. We designed and synthesized peptoid dimers incorporating a disulfide bond and screened their biological activities to evaluate the dimeric effect. Furthermore, to distinguish the impact of the dimerization strategy, we prepared hydroxyl-containing peptoid monomers and compared them with thiol-containing peptoid monomers and their disulfide-linked dimer forms. Dimerization was achieved using both air oxidation and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) for irreversible linkage. Circular dichroism (CD) spectroscopy confirmed the structures, while comprehensive screening (antimicrobial activity, hemolysis, and cytotoxicity) established the influence of dimeric architecture on the overall pharmacological profiles in peptoids. Collectively, this work highlights comprehensive strategies for antimicrobial peptoid discovery, investigating multitarget mechanisms of peptoids with mechanistic study via 3D ODT, demonstrating novel structural versatility with dimeric constructs in peptoid chemistry, and achieving practical metabolic control through fluorination with retained pharmacological properties. These findings advance the rational design of potent, selective, and pharmacokinetically optimized peptoids critical for combating MDR pathogens.