Gram-negative bacterial pathogens pose grave dangers in clinical and environmental settings alike. They include foodborne disease pathogens such as E. coli 0157:H7, waterborne pathogens such as Vibrio cholera, vector-borne pathogens such as Yersinia pestis, and a disturbingly large number of Gram-negative pathogens that now display resistance to multiple antimicrobial agents.1 Resistant Gram-negative pathogens include some of the most common causes of healthcare-associated infections (HAIs): E. coli (causing urinary tract infections), Acinetobacter baumanii (causing wound infections), Pseudomonas aeruginosa (responsible for bloodstream infections and pneumonia), and Klebsiella pneumoniae (causing various HAIs including pneumonia, urinary tract infections, and bloodstream infections). Multi-drug resistant (MDR) strains of Neisseria gonorrhoeae have been reported (responsible for the sexually transmitted disease gonorrhea, which is the second most commonly reported infectious disease in the United States).1
An estimate of the prevalence of infections due to MDR gram-negative bacilli indicated profound consequences for patient mortality, hospital length of stay, and increased hospital costs.2 By 2050, it has been estimated that multidrug resistance (in total) will be responsible for 300 million deaths and drain up to $100 trillion from the world’s gross domestic product, and gram-negative bacteria are drivers of this trend.3 A recent study of 891 hospitalized patients at one institution with bloodstream infections caused by Gram-negative bacteria indicated that one-third were infected with MDR strains, and this subgroup of infections primarily accounted for increased mean inpatient costs related to bloodstream infections.4
The peculiar structure of the outer membrane is a key reason that so many antimicrobial agents are ineffective against Gram-negative bacteria. The double membrane of these microbes is asymmetrical, containing phospholipids in the inner leaflet and a complex glycolipid called LPS in the outer leaflet. Between them is the aqueous periplasm containing a thin layer of peptidoglycan. The journey of LPS to its final destination at the outer membrane involves being “flipped” across the inner membrane by an ABC transporter protein called MsbA in an ATP-driven transport process.5 As a result, MsbA is critical for virulence, and loss of MsbA function causes rapid membrane disruption and cell death.6
Reasoning that MsbA is a valuable potential antibacterial target, a group of researchers at Genentech screened a library of approximately 3 million small molecules for specific inhibitors of Escherichia coli MsbA7 The researchers also used the Transcreener ADP2 FP ATPase Assay from BellBrook Labs to determine the IC50 of MsbA inhibitors. The initial discovery of quinolone compound G592 led to the identification of more potent compounds G247 (IC50 of 5 nM) and G907 (IC50 of 18 nM).7 The team was able to obtain crystals of MsbA in complex with G907 at 2.9 Å resolution, yielding valuable structural data.7
It turns out that G907 traps MsbA in an inward-facing, LPS-bound conformation by wedging into an evolutionarily conserved transmembrane pocket.7 Additionally, a second mechanism of inhibition by G907 happens due to its effect on critical nucleotide-binding domains (NBDs).7
This dual mode of MbsA antagonism is tantalizing, suggest the authors, because it indicates a possible path toward novel antimicrobial compounds that are desperately needed in the global fight against multidrug-resistance pathogens. “Considering the architectural conservation of the G907-binding pocket in other ABC transporters, this work should enable the discovery of selective antagonists across the broader ABC transporter superfamily,” they write.7
Discovering and evaluating novel ABC transporter inhibitors such as G907 requires robust high-throughput assays that are capable of enabling screens of millions of molecules at high sensitivity. Given the economic toll and loss of life that Gram-negative pathogens cause in the inpatient setting alone, a strong arsenal of new antimicrobials is urgently needed, placing a priority on such research efforts.
- Robyn M. Perrin, PhD