Analysis of Orthogonal Efflux and Permeation Properties of Compounds Leads to the Discovery of New Efflux Pump Inhibitors

Abstract

Optimization of compound permeation into Gram-negative bacteria is one of the most challenging tasks in the development of antibacterial agents. Two permeability barriers�the passive diffusion barrier of the outer membrane (OM) and active drug efflux�act synergistically to protect cells from the antibacterial action of compounds. In Escherichia coli (E. coli) and relatives, these two barriers sieve compounds based on different physicochemical properties that are defined by their interactions with OM porins and efflux pumps, respectively. In this study, we critically tested the hypothesis that the best substrates and inhibitors of efflux pumps are compounds that can effectively permeate the OM and are available at relatively high concentrations in the periplasm. For this purpose, we filtered a large subset of the ZINC15 database of commercially available compounds for compounds containing a primary amine, a chemical feature known to facilitate the uptake through E. coli general porins. The assembled library was screened by ensemble docking to AcrA, the periplasmic component of the AcrAB-TolC efflux pump, followed by experimental testing of the top predicted binders for antibacterial activities, efflux recognition, and inhibition. We found that the filtered primary amine library is a rich source of compounds with effluxinhibiting activities and identified efflux pump inhibitors with novel chemical scaffolds effective against E. coli AcrAB-TolC and efflux pumps of multidrug-resistant clinical isolates of Acinetobacter baumannii. However, primary amines are not required for the recognition of compounds by efflux pumps and their efflux-inhibitory activities.

Unveiling Annona Reticulata's Bioactive Arsenal for Enhanced Antibiotic Effects

“…Following incubation, the diameter of these zones is measured, providing a quantitative assessment of the susceptibility of the microorganisms to the tested antimicrobial agents. For antimicrobial potency, Broth microdilution technique assessed antimicrobial potency [7] . This Broth Microdilution Assay with LB Agar is a standardized protocol for determining the minimum inhibitory concentration (MIC) of antimicrobial agents against bacterial cultures.…”

“…For antimicrobial potency, Broth microdilution technique assessed antimicrobial potency. [7] This Broth Microdilution Assay with LB Agar is a standardized protocol for determining the minimum inhibitory concentration (MIC) of antimicrobial agents against bacterial cultures. We used combination of specific antibiotics with our plant extracts to establish antimicrobial synergy.…”

“…[6] Furthermore, multidrug efflux pumps, found in the periplasm of bacterial membranes, expel substances that surpass the OM, repudiating the cell before they can even achieve their prospective zone of action. [7] Despite antibiotic resistance being on the rise, the chance of new treatments remains remote. [6,8,9] The World Health Organization (WHO) highlights the importance of discovering new antimicrobial drugs or developing diverse potentiators/sensitizers of current antibiotics as an alternate option to this challenge.…”

“…Gram‐negative bacteria have several advantages of having a lipopolysaccharide (LPS) outer membrane to develop multidrug resistance by limiting the pervasion of drugs into the bacterial envelope [6] . Furthermore, multidrug efflux pumps, found in the periplasm of bacterial membranes, expel substances that surpass the OM, repudiating the cell before they can even achieve their prospective zone of action [7] . Despite antibiotic resistance being on the rise, the chance of new treatments remains remote [6,8,9] .…”

Efflux Pump-Binding 4(3-Aminocyclobutyl)Pyrimidin-2-Amines Are Colloidal Aggregators

“…However, the replacement of the piperazine ring with a five-or seven-membered ring led to a loss of synergistic activity with erythromycin (23)(24)(25)(26). Likewise, changing the aromatic substituent of the piperidine ring to a heteroaromatic ring (31) or non-cyclic moieties (32)(33)(34) resulted in a loss of synergism. The presence of substituents at the terminal phenyl ring improved the MPC 4 (14) compared to an unsubstituted ring (13).…”