ABSTRACT
BACKGROUND: G protein-coupled receptors (GPCRs) comprise a family of membrane proteins that can be activated by a variety of external factors. The µ-opioid receptor (MOR), a class A GPCR, is the main target of morphine. Recently, enhanced sampling molecular dynamics simulations of a constitutively active mutant of MOR in its apo form allowed us to capture the novel intermediate states of activation, as well as the active state. This prompted us to apply the same techniques to wild type MOR in complex with ligands, in order to explore their contributions to the receptor conformational changes in the activation process. METHODS: MOR was modeled in complex with agonists (morphine, BU72), a partial agonist (naloxone benzoylhydrazone) and an antagonist (naloxone). Replica exchange with solute tempering (REST2) molecular dynamics simulations were carried out for all systems. Trajectory frames were clustered, and the activation state of each cluster was assessed by two different methods. RESULTS: Cluster sizes and activation indices show that while agonists stabilized structures in a higher activation state, the antagonist behaved oppositely. Morphine tends to drive the receptor towards increasing R165-T279 distances, while naloxone tends to increase the NPxxYA motif conformational change. CONCLUSIONS: Despite not observing a full transition between inactive and active states, an important conformational change of transmembrane helix 5 was observed and associated with a ligand-driven step of the process. GENERAL SIGNIFICANCE: The activation process of GPCRs is widely studied but still not fully understood. Here we carried out a step forward in the direction of gaining more details of this process.
Subject(s)
Amino Acids/chemistry , Apoproteins/chemistry , Morphine/chemistry , Phosphatidylcholines/chemistry , Receptors, Opioid, mu/chemistry , Amino Acids/metabolism , Apoproteins/metabolism , Binding Sites , Humans , Ligands , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Morphinans/chemistry , Morphinans/metabolism , Morphine/metabolism , Naloxone/analogs & derivatives , Naloxone/chemistry , Naloxone/metabolism , Phosphatidylcholines/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Pyrroles/chemistry , Pyrroles/metabolism , Receptors, Opioid, mu/metabolism , Solutions , Water/chemistry , Water/metabolismABSTRACT
Chalcones are α,ß-unsaturated ketones containing the 1,3-diarylprop-2-en-1-one framework. This study aims to evaluate the potentiation of antibacterial activity by the chalcone (E)-1-(4-aminophenyl)-3-(furan-2-yl)-prop-2-en-1-one (C13H11NO2), hereafter named AFPO, against multi-resistant strains of Staphylococcus aureus and Escherichia coli. AFPO was synthesized using the Claisen-Schmidt condensation reaction, and the molecular structure was confirmed by nuclear magnetic resonance (NMR). The antibacterial and potentiating properties of AFPO were evaluated by measuring the minimum inhibitory concentration (MIC) using microdilution plates. The AFPO MIC was 1024 µg/mL for the S. aureus 10 strain, revealing synergy in combination with the following antibiotics: penicillin, norfloxacin, ampicillin/sulbactam, and gentamicin. The AFPO MIC was 256 µg/mL for the E. coli 06 strain, and synergy was observed with norfloxacin, gentamicin, and penicillin. The potentiation of antibacterial activity by AFPO was observed against the strains of S. aureus 10 and E. coli 06.
Subject(s)
Chalcone , Chalcones , Escherichia coli Proteins , Symporters , Anti-Bacterial Agents/pharmacology , Chalcone/pharmacology , Chalcones/pharmacology , Escherichia coli , Furans , Microbial Sensitivity Tests , Staphylococcus aureusABSTRACT
G-protein coupled receptors (GPCRs) are the largest and most pharmaceutically relevant family of membrane proteins. Here, fully unbiased, enhanced sampling simulations of a constitutively active mutant (CAM) of a class A GPCR, the µ-opioid receptor (µOR), demonstrates repeated transitions between the inactive (IS) and active-like (AS-L) states. The interconversion features typical activation/inactivation patterns involving established conformational rearrangements of conserved residues. By contrast, wild-type µOR remains in IS during the same course of simulation, consistent with the low basal activity of the protein. The simulations point to an important role of residue W2936.48 at the "toggle switch" in the mutation-induced constitutive activation. Such role has been already observed for other CAMs of class A GPCRs. We also find a significantly populated intermediate state, rather similar to IS. Based on the remarkable accord between simulations and experiments, we suggest here that this state, which has escaped so far experimental characterization, might constitute an early step in the activation process of the apo µOR CAM.
Subject(s)
Apoproteins/chemistry , Mutation , Receptors, Opioid, mu/chemistry , Humans , Models, Molecular , Protein Conformation , Receptors, Opioid, mu/geneticsABSTRACT
The compound 4-(Phenylsulfonyl) morpholine belongs to the class of sulfonamides, which are widely used in the treatment of a large number of diseases caused by microorganisms. This compound has a morpholine group, which is also known for its antimicrobial properties. The aim of the present study was to investigate the antimicrobial and modulating activity of 4-(Phenylsulfonyl) morpholine against standard and multi-resistant strains of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and strains of the fungi Candida albicans, C. tropicalis and C. krusei. Antimicrobial activity was assessed based on the minimum inhibitory concentration (MIC) using the microdilution method. MIC was ⩾1024 µg/mL for all microorganisms. Regarding modulating activity, the most representative effect occurred with the combination of 4-(Phenylsulfonyl) morpholine at a concentration of 128 µg/mL (MIC 1/8) and amikacin against P. aeruginosa 03, with a reduction in MIC from 312.5 to 39.06 µg/mL.
ABSTRACT
The n-hexane extract of the stem bark of Croton anisodontus yielded 2-hydroxy-3,4,6-trimethoxyacetophenone, a well-known substance, but isolated from this species for the first time. The antimicrobial and modulatory activities of the compound towards Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, C. krusei and C. tropicalis strains were assessed. Antibiotics such as amikacin, gentamicin and neomycin were used in a sub-inhibitory concentration. Significant activity was observed towards P. aeruginosa and S. aureus 358, with p < 0.001 in association with amikacin. The present results place C anisodontus as an alternative source of 2-hydroxy-3,4,6-trimethoxyacetophenone with antibacterial potential.
