Beyond Amphiphilic Balance: Changing Subunit Stereochemistry Alters the Pore-Forming Activity of Nylon-3 Polymers

Abstract

Amphiphilic nylon-3 polymers have been reported to mimic the biological activities of natural antimicrobial peptides, with high potency against bacteria and minimal toxicity toward eukaryotic cells. Amphiphilic balance, determined by the proportions of hydrophilic and lipophilic subunits, is considered one of the most important features for achieving this activity profile for nylon-3 polymers and many other antimicrobial polymers. Insufficient hydrophobicity often correlates with weak activities against bacteria, whereas excessive hydrophobicity correlates with high toxicity toward eukaryotic cells. To ask whether factors beyond amphiphilic balance influence polymer activities, we synthesized and evaluated new nylon-3 polymers with two stereoisomeric subunits, each bearing an ethyl side chain and an aminomethyl side chain. Subunits that differ only in stereochemistry are predicted to contribute equally to amphiphilic balance, but we observed that the stereochemical difference correlates with significant changes in biological activity profile. Antibacterial activities were not strongly affected by subunit stereochemistry, but the ability to disrupt eukaryotic cell membranes varied considerably. Experiments with planar lipid bilayers and synthetic liposomes suggested that eukaryotic membrane disruption results from polymer-mediated formation of large pores. Collectively, our results suggest that factors other than amphiphilic balance influence the membrane activity profile of synthetic polymers. Subunits that differ in stereochemistry are likely to have distinct conformational propensities, which could potentially lead to differences in the average shapes of polymer chains, even when the subunits are heterochiral. These findings highlight a dimension of polymer design that should be considered more broadly in efforts to improve specificity and efficacy of antimicrobial polymers.

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“…59 Their results showed that replacing a small portion of cationic or hydrophobic residues with neutral-polar residues had a minor inuence on antimicrobial potency, but decreased toxicity signicantly. Beyond amphiphilicity, the stereochemistry of the monomers signicantly inuenced toxicity towards eukaryotic cells, 60 which may be related to the different conformational propensities of different enantiomeric monomers: ME-cis: TM (Fig. 2b(6)) with extended conformations showed less toxicity to erythrocytes than the corresponding b-peptides with transconformation.…”

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“…[42][43][44] In analogy, LUVs made of a mixture of 90% of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) as zwitterionic lipid with 10% of 1,2-dioleoyl-sn-glycero-3phospho-L-serine (DOPS) which is an anionic lipid, were used as a proxy for red blood cell membranes. [44,45] Calcein, here For liposomes mimicking the membrane of E. coli, both S40…”

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“…For example, a study by Gellman et al found that changes in the polymer subunit stereochemistry alter the activity of nylon-3 copolymers [89]; in particular, stereoisomeric monomers bearing either a cis-aminomethyl side chain or a trans-aminomethyl side chain were created and tested for biological activity. The two stereochemical derivatives had an insignificant influence on the antimicrobial activity but demonstrated significant differences in hemolysis (Figure 4a) [89]. McBride et al [90] also described a series of cationic homopolymers comprising only cationic subunits with strong efficacy against vegetative and spore forms of C. difficile.…”

“…A study by Boyer C et al [91] concluded that by varying the combinations and order of polymeric blocks, the Beyond amphiphilic balance, several studies have revealed exciting results that could help re-formulate the way antimicrobial polymers are designed to improve activity. For example, a study by Gellman et al found that changes in the polymer subunit stereochemistry alter the activity of nylon-3 copolymers [89]; in particular, stereoisomeric monomers bearing either a cis-aminomethyl side chain or a trans-aminomethyl side chain were created and tested for biological activity. The two stereochemical derivatives had an insignificant influence on the antimicrobial activity but demonstrated significant differences in hemolysis (Figure 4a) [89].…”