The eNTRy rules are empirical guidelines that describe physicochemical features enabling small molecules to enter and accumulate in Gram-negative bacteria. They are derived from LC MS/MS-based uptake studies of selected compounds in E. coli.ref
The rules state that compounds are most likely to accumulate when they
- contain a non-sterically encumbered ionizable Nitrogen (primary amines preferred). This feature facilitates protonation under physiological conditions, aiding in membrane penetration.
- have low Three-dimensionality (globularity value ≤0.25. This flatness enhances the likelihood of passive diffusion across the bacterial membrane.
- are relatively Rigid (typically ≤5 rotatable bonds), which improves membrane permeability.
- exhibit balanced amphiphilicity (a balance between hydrophobic and hydrophilic regions). Hydrophobicity aids in membrane interaction, but excessive lipophilicity can hinder permeation.
These rules, while not hard filters, provide a general framework that may help medicinal chemists predict and design compounds with improved penetration and accumulation in Gram-negative bacteria., While these rules can guide the modification of Gram-positive-only scaffolds into analogues with Gram-negative activity, such modifications do not guarantee Gram-negative activity, as not all compounds will follow these patterns, and exceptions are common.
These rules have been operationalized through a web tool (“eNTRyway”) and validated across multiple chemotypes.