Toward Screening for Antibiotics with Enhanced Permeation Properties through Bacterial Porins

Abstract

Gram-negative bacteria are protected by an outer membrane barrier, and to reach their periplasmic target, penicillins have to diffuse through outer membrane porins such as OmpF. Here we propose a structure-dynamics-based strategy for improving such antibiotic uptake. Using a variety of experiments (high-resolution single channel recording, Minimum Inhibitory Concentration (MIC), liposome swelling assay) and accelerated molecular simulations, we decipher the subtle balance of interactions governing ampicillin diffusion through the porin OmpF. This suggests mutagenesis of a hot spot residue of OmpF for which additional simulations reveal drastic changes in the molecular and energetic pathway of ampicillin's diffusion. Inverting the problem, we predict and describe how benzylpenicillin diffuses with a lower effective energy barrier by interacting differently with OmpF. The thorough comparison between the theoretical predictions and the three independent experiments, which were set up to measure the kinetics of transport and biological activity, gives insights on how to combine such different investigation techniques with the aim of providing complementary validation. Our study illustrates the importance of microscopic interactions at the constriction region of the biological channel to control the antibiotic flux through it. We conclude by providing a complete inventory of the channel and antibiotic hot spots and discuss the implications in terms of antibacterial screening and design.

Permeation rates of penicillins indicate that Escherichia coli porins function principally as nonspecific channels

“…1). This is perhaps not surprising, because the affinity of AMP to the binding site is extremely weak, with the dissociation constant reported as 1.9 M (by planar bilayer assay) (6) and 1 M (by molecular dynamics simulation) (10). Thus, in the range of concentrations we used (less than 30 μM for AMP), the drug was at least four orders of magnitude below its K D .…”

“…There are two classes for these models. Some workers assume that permeation of a drug (such as AMP) is retarded by the specific binding (10). Such a model predicts that drugs that do not bind to the binding site (such as PEN) will permeate more rapidly than those that bind (such as AMP).…”

High Salt Concentrations Increase Permeability through OmpC Channels of Escherichia coli

“…Another alternative explanation is based on the specific binding of Amp to the interior of the OmpF channel (36,37). However, this is most unlikely because PEN, which has not been shown to interact strongly with the channel, behaves in a similar manner, and most importantly a similar difference between OmpC and OmpF, as well as the salt-induced enhancement through OmpC, was also seen with lactose.…”

Physical methods to quantify small antibiotic molecules uptake into Gram-negative bacteria

“…These variables, which functional combination can be defined as the approximated reaction coordinate, are biased to allow an efficient tough not complete exploration of the huge space of all possible conformations (ergodic hypothesis). The results obtained within these two approximations, the reduced space and the lack of a real ergodicity, can help only when complemented with experimental results coming from different techniques, such as electrophysiology, liposome swelling assays, enzymatic assay [33,34]. The use of modelling methods in supporting/clarifying experiments are becoming popular in different fields thanks also to the development of supercomputer centres accessible through national and international grants (see in Europe the PRACE consortium, http://www.prace-ri.eu/) and very recently to the use of cheaper GPU for accelerating calculations on local computer resources [35].…”