A kinetic Monte Carlo approach to investigate antibiotic translocation through bacterial porins (original) (raw)
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Biophysical journal, 2010
Our aim in this study was to provide an atomic description of ampicillin translocation through OmpF, the major outer membrane channel in Escherichia coli and main entry point for b-lactam antibiotics. By applying metadynamics simulations, we also obtained the energy barriers along the diffusion pathway. We then studied the effect of mutations that affect the charge and size at the channel constriction zone, and found that in comparison to the wild-type, much lower energy barriers are required for translocation. The expected higher translocation rates were confirmed on the macroscopic scale by liposome-swelling assays. A microscopic view on the millisecond timescale was obtained by analysis of temperature-dependent ion current fluctuations in the presence of ampicillin and provide the enthalpic part of the energy barrier. By studying antibiotic translocation over various timescales and length scales, we were able to discern its molecular mechanism and rate-limiting interactions, and draw biologically relevant conclusions that may help in the design of drugs with enhanced permeation rates.
Microscopic mechanism of antibiotics translocation through a porin
OmpF from the outer membrane of Escherichia coli is a general porin considered to be the main pathway for b-lactam antibiotics. The availability of a high-resolution crystal structure of OmpF and new experimental techniques at the singlemolecule level have opened the way to the investigation of the microscopic mechanisms that allow the passage of antibiotics through bacterial pores. We applied molecular dynamics simulations to investigate the translocation process of ampicillin (Amp) through OmpF. Using a recent algorithm capable of accelerating molecular dynamics simulations we have been able to obtain a reaction path for the translocation of Amp through OmpF. The mechanism of passage depends both on the internal degrees of freedom of Amp and on interactions of Amp with OmpF. Understanding this mechanism would help us design more efficient antibiotics and shed light on nature's way of devising channels able to enhance the transport of molecules through membranes.
Journal of Molecular Modeling, 2010
In Gram-negative bacteria, outer-membrane protein channels, such as OmpF of Escherichia coli, constitute the entry point of various classes of antibiotics. While antibacterial research and development is declining, bacterial resistance to antibiotics is rising and there is an emergency call for a new way to develop potent antibacterial agents and to bring them to the market faster and at reduced cost. An emerging strategy is to follow a bottom-up approach based on microscopically founded computational based screening, however such strategy needs better-tuned methods. Here we propose to use molecular dynamics (MD) simulations combined with the metadynamics algorithm, to study antibiotic translocation through OmpF at a molecular scale. This recently designed algorithm overcomes the time scale problem of classical MD by accelerating some reaction coordinates. It is expected that the initial assumption of the reaction coordinates is a key determinant for the efficiency and accuracy of the simulations. Previous studies using different computational schemes for a similar process only used one reaction coordinate, which is the directionality. Here we go further and see how it is possible to include more informative reaction coordinates, accounting explicitly for: (i) the antibiotic flexibility and (ii) interactions with the channel. As model systems, we select two compounds covering the main classes of antibiotics, ampicillin and moxifloxacine. We decipher the molecular mechanism of translocation of each antibiotic and highlight the important parameters that should be taken into account for improving further simulations. This will benefit the screening and design for antibiotics with better permeation properties.
The Journal of Physical Chemistry B, 2010
We use a multiscale approach, combining molecular dynamics simulations with metadynamics, to simulate the translocation of ampicillin through OmpF from Escherichia coli (E. coli). In-depth analysis has allowed us to reveal the complete picture of the translocation process in terms of both energetics and physicochemical properties. We have demonstrated the existence of a unique affinity site at the constriction region, accessible from both sides and defined by specific pore-antibiotic interactions. By providing optimal binding, the constriction region works like an enzyme toward the permeation of ampicillin. We find reduction in entropy to be compensated by enthalpic contributions from a favorable network of interactions (hydrogen bonds and hydrophobic contacts) which is also mediated by two slow water molecules bridging the antibiotic-pore interactions. Finally, as ampicillin assumes a preferential value for a torsional angle when at the constriction region, we investigated the consequence of the conformational preorganization of ampicillin toward its translocation. As a whole, our analysis opens the way to chemical modifications of antibiotics to allow improving uptake through porins contributing to combat bacterial resistance.
The emergence of multi-drug resistant pathogens led to a critical need for new antibiotics. A key property of effective antibiotics against Gram-negative bacteria is their ability to permeate through the bacterial outer membrane via transmembrane porin proteins. Molecular dynamics (MD) simulations are in principle capable of modeling antibiotic permeation across outer membrane porins (OMPs). However, owing to sampling problems, it has remained challenging to obtain converged potentials of mean force (PMFs) for antibiotic permeation across OMPs. Here, we investigated the convergence of PMFs obtained with three advanced flavors of the umbrella sampling (US) technique aimed to quantify the permeation of the antibiotic fosmidomycin across the OprO porin: (i) Hamiltonian replica-exchange with solute tempering in combination with US, (ii) simulated tempering-enhanced US, and (iii) replica-exchange US. To quantify the PMF convergence and to reveal hysteresis problems, we computed several i...
Toward Screening for Antibiotics with Enhanced Permeation Properties through Bacterial Porins
Biochemistry, 2010
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 structuredynamics-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.
Biophysical Journal, 2011
We have studied the dynamics of chloride and potassium ions in the interior of the OmpF porin under the influence of an external electric field. From the results of extensive all-atom molecular dynamics simulations of the system we computed several first passage time (FPT) quantities to characterize the dynamics of the ions in the interior of the channel. Such FPT quantities obtained from MD simulations demonstrate that it is not possible to describe the dynamics of chloride and potassium ions inside the whole channel with a single constant diffusion coefficient. However, we showed that a valid, statistically rigorous, description in terms of a constant diffusion coefficient D and an effective deterministic force F eff can be obtained after appropriate subdivison of the channel in different regions suggested by the X-ray structure. These results have important implications for popular simplified descriptions of channels based on the 1D Poisson-Nernst-Planck (PNP) equations. Also, the effect of entropic barriers on the diffusion of the ions is identified and briefly discussed.
Simulation of Transport Processes of Antibiotics
2000
In recent years, it has become increasingly apparent that many of the methods used in the field of chemical engineering are applicable to the problems of living systems. In the pharmacokinetic practice, it is important to know the concentration-time profile of antibiotics in tissues, since most infections start and progress outside the vascular bed. Complex processes acting simultaneously and interdependently determine drug distribution. These problems may be solved through the application of modern engineering methods. Many mathematical models have been proposed in the medical and chemical engineering literature, for describing the transport and distribution of antibiotics. The passive diffusion is the most important transport mode of antibiotics in lungs. In the present paper, a simulation study of the Cefazolin penetration in the pulmonary tissue of rabbits, on the basis of a diffusional model, is reported. The computation of the concentration-time profiles of Cefazolin i.v. administrated in the lung parenchyma, could be important for prediction of the therapeutic efficacy of the antibiotic treatment in bacterial respiratory tract infections.
Physical Review E, 2011
We have studied the dynamics of chloride and potassium ions in the interior of the Outer membrane porin F (OmpF) under the influence of an external electric field. From the results of extensive all-atom molecular dynamics (MD) simulations of the system, we computed several first-passage-time (FPT) quantities to characterize the dynamics of the ions in the interior of the channel. Such FPT quantities obtained from MD simulations demonstrate that it is not possible to describe the dynamics of chloride and potassium ions inside the whole channel with a single constant diffusion coefficient. However, we showed that a valid, statistically rigorous description in terms of a constant diffusion coefficient D and an effective deterministic force Feff can be obtained after appropriate subdivison of the channel in different regions suggested by the x-ray structure. These results have important implications for popular simplified descriptions of channels based on the one-dimensional Poisson-Nernst-Planck equations. Also, the effect of entropic barriers on the diffusion of the ions is identified and briefly discussed.
Journal of Biomolecular Screening, 2010
1 School of engineering and Science, Jacobs university bremen, bremen, germany. 2 nanion technologies gmbh, munich, germany. a chip-based automated patch-clamp technique provides an attractive biophysical tool to quantify solute permeation through membrane channels. Proteo-giant unilamellar vesicles (proteo-guvs) were used to form a stable lipid bilayer across a micrometer-sized hole. because of the small size and hence low capacitance of the bilayer, single-channel recordings were achieved with very low background noise. the latter allowed the characterization of the influx of 2 major classes of antibioticscephalosporins and fluoroquinolones-through the major Escherichia coli porins ompf and ompc. analyzing the ion current fluctuations in the presence of antibiotics revealed transport properties that allowed the authors to determine the mode of permeation. the chip-based setup allows rapid solution exchange and efficient quantification of antibiotic permeation through bacterial porins on a single-molecule level. (Journal of Biomolecular Screening 2010:302-307)