Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA

Abstract

There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

Benzimidazolium salts prevent and disrupt methicillin-resistant Staphylococcus aureus biofilms

“…Gram-negative bacteria possess an outer membrane acting as an extra protective layer that usually reduces the antibacterial power of synthetic compounds. 25 Our new analogues may act on the outer cellular membrane inducing its permeabilization. In addition to their interesting antibacterial activity, these new analogues show low toxicity on red blood cells ( Table 1, HC 50 values).…”

“…The reaction was allowed to warm up to R.T. and stirred until no more starting benzimidazole was observed by TLC (100% EtOAc) (2-3 hours). The reaction was then quenched with water and the aqueous layer was extracted three times with EtOAc, dried over Na 2 SO 4 and puried using silica gel chromatography (100% EtOAc) to afford the corresponding alkylated benzimidazole(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)). Yield 62%, 1 H NMR (500 MHz, DMSO-d 6 ) d 8.42 (s, 1H), 7.71-7.63 (m, 1H), 7.57-7.46 (m, 3H), 7.29-7.24 (m, 2H), 7.24-7.15 (m, 2H), 5.49 (s, 2H).…”

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A

“…Indeed, the lack of a functional MsbA flippase causes growth inhibition, lipid A accumulation in the IM with appearance of vesicle- or tubule-like shapes, and membrane stacks. Moreover, MsbA-depleted cells show enlarged size [ 55 , 56 ]. In addition, Lpt complex depletion has a bacteriostatic effect accompanied by moderate lysis and formation of short filaments.…”

Folded Synthetic Peptides and Other Molecules Targeting Outer Membrane Protein Complexes in Gram-Negative Bacteria

“…This leads to elevated levels of LPS at the IM, which has a deleterious effect on the membrane. In a different study, a high throughput in-vitro assay with purified MsbA led to the identification of a family of quinoline derivatives that specifically inhibit the activity of MsbA on cells (Figure 2C; Alexander et al, 2018; Ho et al, 2018). These molecules typically inhibit the growth of E. coli and Klebsiella pneumoniae with MICs in the low micromolar range.…”

“…Crystal structures were also obtained of E. coli MsbA with bound quinoline inhibitors, which show the protein trapped in an inward-facing LPS-bound conformation. Unfortunately, the hydrophobic nature of these active quinolines correlated with high levels of plasma protein binding and a significant loss of antimicrobial activity in the presence of serum, indicating non-optimal drug-like properties (Alexander et al, 2018). Nevertheless, these studies validate MsbA as an antibacterial target and establish screening methods that can be used to discover new LPS transport inhibitors.…”