General Method to Determine the Flux of Charged Molecules through Nanopores Applied to β-Lactamase Inhibitors and OmpF

Abstract

A major challenge in the discovery of the new antibiotics against Gram-negative bacteria is to achieve sufficiently fast permeation in order to avoid high doses causing toxic side effects. So far, suitable assays for quantifying the uptake of charged antibiotics into bacteria are lacking. We apply an electrophysiological zero-current assay using concentration gradients of β-lactamase inhibitors combined with single-channel conductance to quantify their flux rates through OmpF. Molecular dynamic simulations provide in addition details on the interactions between the nanopore wall and the charged solutes. In particular, the interaction barrier for three β-lactamase inhibitors is surprisingly as low as 3-5 kcal/mol and only slightly above the diffusion barrier of ions such as chloride. Within our macroscopic constant field model, we determine that at a zero-membrane potential a concentration gradient of 10 μM of avibactam, sulbactam, or tazobactam can create flux rates of roughly 620 molecules/s per OmpF trimer.

The characteristics of antibiotic resistance and phenotypes in 29 outer‐membrane protein mutant strains in Aeromonas hydrophila

“…However, toxins and antibiotics are also small molecules that might enter into the bacterial cell through the OMPs as well, which caused bacteria to evolve many tactics to obtain antibiotic resistance characteristics by turning off the antibiotics channel or boost the pump activity of the OMPs (Delcour, ; Ghai and Ghai ; Ghai and Ghai ). For example, OmpC and OmpF are two major OMPs in Escherichia coli that act as passive diffusion channels for small molecules, such as nutrients, toxic salts and antibiotics (Liu et al ., ; Ghai et al ., ). Both proteins are controlled by the OmpR/EnvZ and CpxR/CpxA two‐component regulatory systems and display opposite behaviours when facing different kinds of antibiotics (Lin et al ., ).…”

Fosfomycin Permeation through the Outer Membrane Porin OmpF

“…In general, the transport of antibiotic molecules, especially across the OM of Gram-negative bacteria, has been identified as one of the key problems in antibiotics research (14)(15)(16). Recently, considerable effort-both experimentally as well as theoretically-has been put into this subject (see, for example, (17)(18)(19)(20)(21)(22)(23)(24)(25)), which is quite complex, partially because of the large diversity of channels and the so far often-incomplete knowledge of their structure and functional properties.…”

High-resolution experimental and computational electrophysiology reveals weak β-lactam binding events in the porin PorB

“…The binding site revealed by our approach is wedged between the clusters of conserved acidic and basic residues of the eyelet region, suggesting a common binding site for zwitterionic drugs within porin channels, in which ligand binding is strongly determined by the transverse electrostatic field. 32,43 Importantly, we were able to use information on a marked asymmetry in the experimental currents across the drug-bound channel, which is often detected in ligand-bound membrane pores, and which has been shown to be caused by the electro-osmotic flow of water through the channel, 33,34,43,48 to validate the binding mode and characterise the drug-channel interaction in detail. These results show that a combination of well-established techniques with new analysis protocols permits the identification and validation of previously undetectable low-affinity drug binding events in a biological channel protein.…”

“…4,31 In order to provide structural information at sufficient detail for improved drug design, 19,32 it is important to reliably identify these scenarios, and to characterise the molecular determinants of any potential binding event between the antibiotic molecule and the protein channel. 33,34 Here we demonstrate how we have combined new and traditional methodologies of membrane channel characterisation to achieve this.…”