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

Abstract

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.

Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores

“…[1][2][3] One of the current bottlenecks in the development of molecules active against Gram-negative bacteria is their low permeability across the bacterial cell wall, composed of an outer membrane (OM) and inner membrane (IM). 4,5 Methods to investigate the accumulation of antimicrobial agents inside a cell can be in vivo viability assay, 6 liposome swelling assay, 7 mass spectrometry and uorometry related approaches, [8][9][10] and electrophysiology. 5,11 Nanopore based single molecule tools could already investigate a single nucleotide segment, 12,13 and nucleotide base lesion analogues, 14,15 peptides [16][17][18][19] etc.…”

Two distinct mechanisms of small molecule inhibition of LpxA acyltransferase essential for lipopolysaccharide biosynthesis

“…In addition to affinity enhancement, compound 2 series need further improvement in the MICs against clinical isolates of E. coli (and other Gram-negative pathogens) in order to move the LpxA inhibitor forward on lead optimization. In general, understanding the chemical properties that facilitate compound accumulation in Gram-negative bacterial cells by overcoming outer membrane permeability and efflux is important for antibacterial discovery 4, 51–53 . However, universal guidelines that can be used to optimize compounds for better bacterial cell accumulation are not yet established because molecular descriptors for compound accumulation vary for each chemical scaffold 53 .…”

“…In general, understanding the chemical properties that facilitate compound accumulation in Gram-negative bacterial cells by overcoming outer membrane permeability and efflux is important for antibacterial discovery 4, 51–53 . However, universal guidelines that can be used to optimize compounds for better bacterial cell accumulation are not yet established because molecular descriptors for compound accumulation vary for each chemical scaffold 53 . Understanding of cell entry for this chemical series as well as insights into increasing binding-affinity to the LpxA-product complex are necessary to further increase potency and accumulation in E. coli .…”

Distribution of fluoroquinolones in the two aqueous compartments of Escherichia coli

“…The importance of cellular accumulation to any pharmaceuticals is apparent. Several studies have been conducted to determine the correlation between antibiotic accumulation in bacteria and its MIC [ [14] , [15] , [16] , [17] , [18] , [19] ]. Bazile et al determined the cellular accumulation of 11 fluoroquinolones and their DNA gyrase inhibition activity [ 18 ].…”

“…In a recent study, Iyer et al determined accumulation of fluoroquinolones and a collection of other DNA ligase inhibitors in E. coli using mass spectroscopy and observed that there was no correlation between accumulation of fluoroquinolones versus their effectiveness in inhibiting bacteria cell growth [ 17 ]. No correlation was observed between accumulation concentration and the ratio of IC 50 to MIC of the DNA ligases.…”