Evaluating LC–MS/MS To Measure Accumulation of Compounds within Bacteria

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 ΔtolC). 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.