High-throughput screening of cationic lipidoids reveals how molecular conformation affects membrane-targeting antimicrobial activity

Abstract

The search for next-generation antibacterial compounds that overcome the development of resistance can be facilitated by identifying how to target the cell membrane of bacteria. Understanding the key molecular features that enable interactions with lipids and lead to membrane disruption is therefore crucial. Here we employ a library of lipid-like compounds (lipidoids) comprising modular structures with tunable hydrophobic and hydrophilic architecture, to shed light on how the chemical functionality and molecular shape of synthetic amphiphilic compounds determine their activity against bacterial membranes. Synthesized from combinations of 8 different polyamines as headgroups and 13 acrylates as tails, 104 different lipidoids are tested for activity against a model Gram-positive bacterial strain (Bacillus subtilis). Results from the high-throughput antimicrobial screening assay show that lipidoids with the most potent antimicrobial properties (down to 2 μM) have intermediate tail hydrophobicity (i.e. logP values between 3 and 4), and lower headgroup charge density (i.e. longer spacer groups between charged amines). However, the most important factor appeared to be the ability of a lipidoid to self-assemble into an inverse hexagonal liquid crystalline phase, as observed by small angle X-ray scattering (SAXS) analysis. The lipidoids active at lowest concentrations, and which induced the most significant membrane damage during propidium iodide (PI) permeabilization assays, were those that aggregated into highly-curved inverse hexagonal liquid crystal phases. These observations suggest that the introduction of strong curvature stress into the membrane is one way to maximize membrane disruption and lipidoid antimicrobial activity. Lipidoids that demonstrated the ability to furnish this phase consisted of either i) branched or linear headgroups with shorter linear tails, or ii) cyclic headgroups with 4 bulky non-linear tails. On the contrary, lipidoids previously observed to adopt disc-like conformations that pack into bicontinuous cubic phases were significantly less effective against B. subtilis. The discovery of these structure-property relationships demonstrates that it is not simply a balance of hydrophobic and hydrophilic moieties that governs membrane-active antibacterial activity, but also their intrinsic curvature and collective behavior.

Bactericidal Activity to Escherichia coli: Different Modes of Action of Two 24-Mer Peptides SAAP-148 and OP-145, Both Derived from Human Cathelicidine LL-37

“…For example, amphibian temporins [15] have a very high hydrophobic content of 69%, and peptides with lower hydrophobic content, such as thrombocydine TC-19 (40%) [16], human cathelicidin LL-37 (40%), and its derivative OP-145 (46%) [13], exhibit slightly higher potency in killing Gram-positive bacteria. It should be noted that the molecular conformation of an antimicrobial, including recently described lipid-like compounds, is a crucial determinant of membrane activity against both Gram-negative and Gram-positive bacteria [30]. However, the general observation suggests that there is a certain degree of permeabilization for zwitterionic membranes, while a clear preference for anionic membranes is often observed.…”