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The drug/proton antiporter AcrB, which is part of the major efflux pump AcrABZ-TolC in Escherichia coli, is the paradigm transporter of the resistance-nodulation-cell division (RND) superfamily. Despite the impressive ability of AcrB to transport many chemically unrelated compounds, only a few of these ligands have been co-crystallized with the protein. Therefore, the molecular features that distinguish good substrates of the pump from poor ones have remained poorly understood to date. In this work, a thorough in silico protocol was employed to study the interactions of a series of congeneric compounds with AcrB to examine how subtle chemical differences affect the recognition and transport of substrates by this protein. Our analysis allowed us to discriminate among different compounds, mainly in terms of specific interactions with diverse sub-sites within the large distal pocket of AcrB. Our findings could provide valuable information for the design of new antibiotics that can evade the antimicrobial resistance mediated by efflux pump machinery.

The major cause of bacterial resistance to β-lactams is the production of hydrolytic β-lactamase enzymes. Nowadays, the combination of β-lactam antibiotics with β-lactamase inhibitors (BLIs) is the main strategy for overcoming such issues. Nevertheless, particularly challenging β-lactamases, such as OXA-48, pose the need for novel and effective treatments. Herein, we describe the screening of a proprietary compound collection against Klebsiella pneumoniae OXA-48, leading to the identification of several chemotypes, like the 4-ideneamino-4H-1,2,4-triazole (SC_2) and pyrazolo[3,4-b]pyridine (SC_7) cores as potential inhibitors. Importantly, the most potent representative of the latter series (ID2, AC50 = 0.99 μM) inhibited OXA-48 via a reversible and competitive mechanism of action, as demonstrated by biochemical and X-ray studies, furthermore, it slightly improved imipenem’s activity in Escherichia coli ATCC BAA-2523 β-lactam resistant strain. Also, ID2 showed good solubility and no sign of toxicity up to the highest tested concentration, resulting in a promising starting point for further optimization programs toward novel and effective non-β-lactam BLIs.

Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [H-3]D-aspartate ([H-3]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release regulating 5-HT2A heteroreceptors. Actually, the 15 mM KCl-evoked [H-3]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HT2A agonist (+/-)DOI, an effect reversed by the 5-HT2A antagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HT2A receptors colocalize and cross-talk in these terminals and if 5-HT2A ligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HT2A receptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HT2A receptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord synaptosomes were also 5-HT2A immunopositive. Interestingly, the 100 pM LY379268-induced reduction of the 15 mM MCI-evoked [H-3]D-Asp overflow as well as its inhibition by 100 nM (+/-)DOI became undetectable when the two agonists were concomitantly added. Conversely, 5-HT2A antagonists (MDL11,939, MDL100907, ketanserin and TZD) reinforced the release-regulating activity of mGlu2/3 autoreceptors. Increased expression of mGlu2/3 receptor proteins in synaptosomal plasmamembranes paralleled the gain of function of the mGlu2/3 autoreceptors elicited by 5-HT2A antagonists. Based on these results, we propose that in spinal cord glutamatergic terminals i) mGlu2/3 and 5-HT2A receptors colocalize and interact one each other in an antagonist-like manner, ii) 5-HT2A antagonists are indirect positive allosteric modulator of mGlu2/3 autoreceptors controlling glutamate exocytosis. (C) 2018 Elsevier Ltd. All rights reserved.

Summary In fragile X syndrome (FXS), CGG repeat expansion greater than 200 triplets is believed to trigger FMR1 gene silencing and disease etiology. However, FXS siblings have been identified with more than 200 CGGs, termed unmethylated full mutation (UFM) carriers, without gene silencing and disease symptoms. Here, we show that hypomethylation of the FMR1 promoter is maintained in induced pluripotent stem cells (iPSCs) derived from two UFM individuals. However, a subset of iPSC clones with large CGG expansions carries silenced FMR1. Furthermore, we demonstrate de novo silencing upon expansion of the CGG repeat size. FMR1 does not undergo silencing during neuronal differentiation of UFM iPSCs, and expression of large unmethylated CGG repeats has phenotypic consequences resulting in neurodegenerative features. Our data suggest that UFM individuals do not lack the cell-intrinsic ability to silence FMR1 and that inter-individual variability in the CGG repeat size required for silencing exists in the FXS population., Graphical Abstract, Highlights • Unmethylated full mutation (UFM) iPSCs and neurons maintain active FMR1 • UFM iPSCs have the capacity to silence FMR1 • CGG repeat size required for silencing in UFM is higher than 200 described for FXS • UFM iPSCs derived neurons show signs of neurodegeneration, Rare individuals do not silence FMR1 and do not develop FXS despite carrying the disease causing CGG expansion. Using iPSCs, Fodor, Di Giorgio, and colleagues show that these individuals have not lost the ability to silence FMR1 but the CGG repeat number required for silencing is higher than the one described for FXS patients.

In this study, a drug discovery programme that sought to identify novel dual bacterial topoisomerase II inhibitors (NBTIs) led to the selection of six optimized compounds. In enzymatic assays, the molecules showed equivalent dual-targeting activity against the DNA gyrase and topoisomerase IV enzymes of Staphylococcus aureus and Escherichia coli. Consistently, the compounds demonstrated potent activity in susceptibility tests against various Gram-positive and Gram-negative reference species, including ciprofloxacin-resistant strains. The activity of the compounds against clinical multidrug-resistant isolates of S. aureus, Clostridium difficile, Acinetobacter baumannii, Neisseria gonorrhoeae, E. coli and vancomycin-resistant Enterococcus spp. was also confirmed. Two compounds (1 and 2) were tested in time-kill and post-antibiotic effect (PAE) assays. Compound 1 was bactericidal against all tested reference strains and showed higher activity than ciprofloxacin, and compound 2 showed a prolonged PAE, even against the ciprofloxacin-resistant S. aureus BAA-1720 strain. Spontaneous development of resistance to both compounds was selected for in S. aureus at frequencies comparable to those obtained for quinolones and other NBTIs. S. aureus BAA-1720 mutants resistant to compounds 1 and 2 had single point mutations in gyrA or gyrB outside of the quinolone resistance-determining region (QRDR), confirming the distinct site of action of these NBTIs compared to that of quinolones. Overall, the very good antibacterial activity of the compounds and their optimizable in vitro safety and physicochemical profile may have relevant implications for the development of new broad-spectrum antibiotics.

Staphylococcus aureus and Staphylococcus epidermidis are leading pathogens of biofilm-related infections and represent the most common cause of osteomyelitis and biomedical implants infections. Biofilm-related infections usually require long-term antibiotic treatment, often associated to surgical interventions. Dalbavancin is a newer lipoglycopeptide approved for the treatment of acute skin and skin-structure infections caused by Gram-positive pathogens. In addition, dalbavancin has recently been considered as a potential option for the treatment of staphylococcal osteomyelitis and orthopedic implant infections. In this study, time-kill kinetics of dalbavancin against S. aureus and S. epidermidis biofilms were determined over prolonged exposure times (up to 7 days), using both a standardized biofilm susceptibility model and biofilms grown onto relevant orthopedic biomaterials (i.e. titanium and cobalt-chrome disks). Dalbavancin (at concentrations achievable in bone and articular tissue) showed a potent activity against established staphylococcal biofilms in both tested models, and was overall superior to the comparator vancomycin.