Cationic Amphiphiles with Specificity against Gram-Positive and Gram-Negative Bacteria: Chemical Composition and Architecture Combat Bacterial Membranes

Abstract

Small-molecule cationic amphiphiles (CAms) were designed to combat the rapid rise in drug-resistant bacteria. CAms were designed to target and compromise the structural integrity of bacteria membranes, leading to cell rupture and death. Discrete structural features of CAms were varied, and structure-activity relationship studies were performed to guide the rational design of potent antimicrobials with desirable selectivity and cytocompatibility profiles. In particular, the effects of cationic conformational flexibility, hydrophobic domain flexibility, and hydrophobic domain architecture were evaluated. Their influence on antimicrobial efficacy in Gram-positive and Gram-negative bacteria was determined, and their safety profiles were established by assessing their impact on mammalian cells. All CAms have a potent activity against bacteria, and hydrophobic domain rigidity and branched architecture contribute to specificity. The insights gained from this project will aid in the optimization of CAm structures.

Figure 1.

Chemical structures of three CAm series indicating nomenclature (bold underlined text) and structural variations. All CAm structures are based on parent CAm (top) with strategic chemical changes. Charge flexibility is varied by increasing the linker length between cationic charges and the sugar backbone (left). Charge and hydrophobic arm flexibility are altered though increasing charged end-group linker lengths in addition to ether-linkages for hydrophobic arms (center). The branched hydrophobic architecture was generated by using dendritic branch points for hydrophobic domains.

Figure 2.

Synthetic approach used to generate CAm-ethers in two reaction steps from T10-ether.

Figure 3.

Synthetic approach used to generate CAm-esters in three reaction steps from readily available starting materials.

Figure 4.

Synthetic approach used to generate branched CAms.

Figure 5.

Cartoon depicting the basic membrane structure of Gram-negative and Gram-positive bacteria. Gram-negative bacteria have a double membrane structure and additional lipopolysaccharide layer compared with Gram-positive bacteria. This figure was adapted from a previous Rutgers thesis.

Figure 6.

Hemolytic activity of CAms. CAm ethers and CAm esters hemolytic potential (left). CAm ethers have filled markers and CAm esters have open markers. Hemolytic activity of branched CAms is shown on the right in addition to hemolytic activity of analogous CAm ether for comparison.

Figure 7.

Cytocompatibility of CAms evaluated against mammalian fibroblasts.

Figure 8.

Calcein leakage experiments of CAm ethers. Leakage of dye from experiments performed with 3a (A) and negatively charged membranes (B) mimicking bacterial cells and neutral membranes (C) mimicking mammalian host cell membranes indicate membrane specificity. A comparison of total dye leakage at the end of the experiment for each membrane type (D) shows influence of CAm concentration.

Figure 9.

Calcein leakage experiments for CAm-esters. Results from experiments performed with 6a (A) and negatively charged membranes (B) mimicking bacterial cells and neutral membranes (C) mimicking mammalian host cell membranes indicate membrane specificity. A comparison of total dye leakage at the end of the experiment for each membrane type (D) shows influence of CAm concentration.