In response to the urgent need for new antibiotic development strategies, antimicrobial peptides (AMPs) and other synthetic polymers are being actively investigated as promising alternatives to traditional antibiotics. Although most AMPs display lytic activity against several types of bacteria, they have poor toxicology profiles and are susceptible to proteolysis in vivo. While many synthetic variants have been created to mimic AMPs by tuning the hydrophobic to cationic ratio of the side-chain groups, few have decoupled the effects of charge from hydrophobicity in discrete systems, and none have investigated the effect of backbone hydrophobicity. We recently developed a rapid and efficient approach for the assembly of synthetic sequence-defined oligothioetheramides (oligoTEAs) that are resistant to protease activity. Our oligoTEA assembly scheme allows direct access to the oligomer backbone, which enables precise tuning of oligoTEA hydrophobicity while keeping charge constant. In this study, we synthesized a new class of antibacterial oligoTEAs (AOTs) with precise control over backbone hydrophobicity and composition. Our studies suggest that AOTs lyse cells via membrane permeabilization and that hydrophobicity and macromolecular conformation are key properties that regulate AOT activity. Some of our AOTs show highly promising antibacterial activity (MIC ∼ 0.5-5 μM) against clinically relevant pathogens in the presence of serum, with little to no toxicity against RBCs and HEK293 cells. Taken together, our data identify design parameters and criteria that may be useful for assembling the next generation of potent and selective AOTs.

Antibiotic discovery has experienced a severe slowdown in terms of discovery of new candidates. In vitro screening methods using phospholipids to model the bacterial membrane provide a route to identify molecules that specifically disrupt bacterial membranes causing cell death. Thanks to the electrically insulating properties of the major component of the cell membrane, phospholipids, electronic devices are highly suitable transducers of membrane disruption. The organic electrochemical transistor (OECT) is a highly sensitive ion-to-electron converter. Here, the OECT is used as a transducer of the permeability of a lipid monolayer (ML) at a liquid:liquid interface, designed to read out changes in ion flux caused by compounds that interact with, and disrupt, lipid assembly. This concept is illustrated using the well-documented antibiotic Polymixin B and the highly sensitive quantitation of permeability of the lipid ML induced by two novel recently described antibacterial amine-based oligothioetheramides is shown, highlighting molecular scale differences in their disruption capabilities. It is anticipated that this platform has the potential to play a role in front-line antimicrobial compound design and characterization thanks to the compatibility of semiconductor microfabrication technology with high-throughput readouts.

In response to the urgent need for new antibiotic development strategies, antimicrobial peptides and their synthetic mimetics are being investigated as promising alternatives to traditional antibiotics. To facilitate their development into clinically viable candidates, we need to understand what molecular features and physicochemical properties are needed to induce cell death. Within the context of sequence-defined oligothioetheramides (oligoTEAs), we explore the impact of the cationic pendant group and backbone hydrophobicity on the potency and selectivity of antibacterial oligoTEAs. Through antibacterial, cytotoxicity, membrane destabilization, and membrane depolarization assays, we find a strong dependency on the nature of the cationic group and improved selectivity toward bacteria by tuning backbone hydrophobicity. In particular, compounds with the guanidinium headgroup are more potent than those with amines. Finally, we identify a promising oligoTEA, PDT-4G, with enhanced activity in vitro (minimum inhibitory concentration (MIC) ∼ 0.78 μM) and moderate activity in a mouse thigh infection model of methicillin-resistant Staphylococcus aureus. The studies outlined in this work provide insights into the effect of macromolecular physicochemical properties on antibacterial potency. This knowledge base will be vital for researchers engaged in the ongoing development of clinically viable antibacterial agents.

Cationic charge and hydrophobicity have long been understood to drive the potency and selectivity of antimicrobial peptides (AMPs). However, these properties alone struggle to guide broad success in vivo, where AMPs must differentiate bacterial and mammalian cells, while avoiding complex barriers. New parameters describing the biophysical processes of membrane disruption could provide new opportunities for antimicrobial optimization. In this work, we utilize oligothioetheramides (oligoTEAs) to explore the membrane-targeting mechanism of oligomers, which have the same cationic charge and hydrophobicity, yet show a unique ~ 10-fold difference in antibacterial potency. Solution-phase characterization reveals little difference in structure and dynamics. However, fluorescence microscopy of oligomer-treated Staphylococcus aureus mimetic membranes shows multimeric lipid aggregation that correlates with biological activity and helps establish a framework for the kinetic mechanism of action. Surface plasmon resonance supports the kinetic framework and supports lipid aggregation as a driver of antimicrobial function.

Biophysical analysis into the mechanism of action of membrane-disrupting antibiotics such as antimicrobial peptides (AMPs) and AMP mimetics is necessary to improve our understanding of this promising but relatively untapped class of antibiotics. We evaluate the impact of cationic nature, specifically the presence of guanidine versus amine functional groups using sequence-defined oligothioetheramides (oligoTEAs). Relative to amines, guanidine groups demonstrated improved antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). To understand the mechanism of action, we evaluated membrane interactions by performing a propidium iodide assay and fluorescence microscopy of supported MRSA mimetic bilayers treated with oligoTEAs. Both studies demonstrated membrane disruption, while fluorescence microscopy showed the formation of lipid aggregates. We further analyzed the mechanism using surface plasmon resonance with a recently developed two-state binding model with loss. Our biophysical analysis points to the importance of lipid aggregation for antibacterial activity and suggests that guanidine groups improve antibacterial activity by increasing the extent of lipid aggregation. Altogether, these results verify and rationalize the importance of guanidines for enhanced antibacterial activity of oligoTEAs, and present biophysical phenomena for the design and analysis of additional membrane-active antibiotics.