A general method for determining bacterial uptake of compounds independent of antibacterial activity would be a valuable tool in antibacterial drug discovery. LC-MS/MS assays have been described, but it has not been shown whether the data can be used directly to inform medicinal chemistry. We describe the evaluation of an LC-MS/MS assay measuring association of compounds with bacteria, using a set of over a hundred compounds (inhibitors of NAD-dependent DNA ligase, LigA) for which in vitro potency and antibacterial activity had been determined. All compounds were active against an efflux-deficient strain of Escherichia coli with reduced LigA activity ( E. coli ligA251 Δ tolC). Testing a single compound concentration and incubation time, we found that, for equipotent compounds, LC-MS/MS values were not predictive of antibacterial activity. This indicates that measured bacteria-associated compound was not necessarily exposed to the target enzyme. Our data suggest that, while exclusion from bacteria is a major reason for poor antibacterial activity of potent compounds, the distribution of compound within the bacterial cell may also be a problem. The relative importance of these factors is likely to vary from one chemical series to another. Our observations provide directions for further study of this difficult issue.
Keywords: Gram-negative; antibacterial; compound accumulation; ligase; mass spectrometry.