Extended Data Fig. 1. Overview of eNTRyway functionality and examples of visual outputs.

eNTRyway classifies molecules that are likely capable of accumulating in E. coli. eNTRyway calculates physiochemical properties of molecules and compares to a training set of compounds. SMILES strings for this analysis are generated in Open Babel using canonicalized atom order. Compounds containing an ionizable nitrogen (primary amines are most favorable), low three-dimensionality (globularity ≤ 0.25), and low flexibility (rotatable bonds ≤ 5) have a significantly higher probability of accumulating in E. coli. It is important to note that localized polarity and sterics about the amine are important considerations when implementing the eNTRy rules as these factors are not explicitly included in the eNTRYway results. Previously, it was found that increased amphiphilic moment (vsurf_A) correlated with increased accumulation while sterically encumbered primary amines displayed decreased accumulation.¹⁴ Using eNTRyway to reanalyze the diverse compound collection published in Richter et al¹⁴ (Tables S2-S4; 188 compounds), we found that the accuracy is 84% (Supplementary Table 6).