New Noncovalent Inhibitors of Penicillin-Binding Proteins from Penicillin-Resistant Bacteria

BACKGROUND: Penicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs beta-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for beta-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs. METHODOLOGY/PRINCIPAL FINDINGS: Our in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains. CONCLUSIONS: We found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.Eur-Intafa

Specific Activation of Estrogen Receptor Alpha and Beta Enhances Male Sexual Behavior and Neuroplasticity in Male Japanese Quail

Two subtypes of estrogen receptors (ER), ERα and ERβ, have been identified in humans and numerous vertebrates, including the Japanese quail. We investigated in this species the specific role(s) of each receptor in the activation of male sexual behavior and the underlying estrogen-dependent neural plasticity. Castrated male Japanese quail received empty (CX) or testosterone-filled (T) implants or were daily injected with the ER general agonist diethylstilbestrol (DES), the ERα-specific agonist PPT, the ERβ-specific agonist DPN or the vehicle, propylene glycol. Three days after receiving the first treatment, subjects were alternatively tested for appetitive (rhythmic cloacal sphincter movements, RCSM) and consummatory aspects (copulatory behavior) of male sexual behavior. 24 hours after the last behavioral testing, brains were collected and analyzed for aromatase expression and vasotocinergic innervation in the medial preoptic nucleus. The expression of RCSM was activated by T and to a lesser extent by DES and PPT but not by the ERβagonist DPN. In parallel, T fully restored the complete sequence of copulation, DES was partially active and the specific activation of ERα or ERβ only resulted in a very low frequency of mount attempts in few subjects. T increased the volume of the medial preoptic nucleus as measured by the dense cluster of aromatase-immunoreactive cells and the density of the vasotocinergic innervation within this nucleus. DES had only a weak action on vasotocinergic fibers and the two specific ER agonists did not affect these neural responses. Simultaneous activation of both receptors or treatments with higher doses may be required to fully activate sexual behavior and the associated neurochemical events

Kinetic properties of the Bacillus licheniformis penicillin-binding proteins.

In the analysis of the interactions between beta-lactam antibiotics and their target enzymes, it is often difficult to estimate the kinetic properties of the molecules which react rapidly with their targets and in consequence behave as the most efficient antibiotics. The combined utilization of fluorescein-labelled penicillins and of a new competition method has allowed an accurate determination of the high second-order rate constants characterizing the acylation of Bacillus licheniformis penicillin-binding protein 1 (PBP1) by penicillins and cephalosporins. Strategies were devised for measuring high acylation rates while avoiding titration effects. The method was also suitable for measuring the PBP kinetic parameters in intact cells. These results also confirmed that PBP1 is probably the main target of most beta-lactam antibiotics. Cephalexin, however, reacted faster with PBP3

A new, highly sensitive method for the detection and quantification of penicillin-binding proteins.

A new method for the identification and quantification of penicillin-binding proteins is described which uses fluorescein-coupled penicillins. It allows the rapid detection of 0.2 pmol with the naked eye and 2 fmol with the help of an A.L.F. automatic DNA sequencer. Direct labelling can also be performed on whole bacterial cells

Synthesis, purification and kinetic properties of fluorescein-labelled penicillins.

The synthesis and properties of six fluorescein-labelled penicillins are reported. The two isomers of fluoresceyl-glycyl-6-amino-penicillanic acid are probably the best compounds to use for detection of all the penicillin-binding proteins (PBPs) present in a bacterial membrane preparation. However, the derivatives of ampicillin were much more efficient against Enterobacter aerogenes PBP3. The two isomers obtained when a commercial mixture of the two isomers of carboxyfluorescein was used most often exhibited similar properties, but the Streptomyces R61 extracellular DD-peptidase was only efficiently acylated by the 5'-carboxyfluorescein derivative of glycyl-6-aminopenicillanic acid

An alternative role of FoF1-ATP synthase in Escherichia coli: synthesis of thiamine triphosphate

In E. coli, thiamine triphosphate (ThTP), a putative signaling molecule, transiently accumulates in response to amino acid starvation. This accumulation requires the presence of an energy substrate yielding pyruvate. Here we show that in intact bacteria ThTP is synthesized from free thiamine diphosphate (ThDP) and Pi, the reaction being energized by the proton-motive force (Dp) generated by the respiratory chain. ThTP production is suppressed in strains carrying mutations in F1 or a deletion of the atp operon. Transformation with a plasmid encoding the whole atp operon fully restored ThTP production, highlighting the requirement for FoF1-ATP synthase in ThTP synthesis. Our results show that, under specific conditions of nutritional downshift, FoF1-ATP synthase catalyzes the synthesis of ThTP, rather than ATP, through a highly regulated process requiring pyruvate oxidation. Moreover, this chemiosmotic mechanism for ThTP production is conserved from E. coli to mammalian brain mitochondria