ETX2514 is a broad-spectrum β-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii.

Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of β-lactamases, enzymes that inactivate β-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new β-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii.

Crystal structure of A. aeolicus LpxC with bound product suggests alternate deacetylation mechanism.

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is the first committed step to form lipid A, an essential component of the outer membrane of Gram-negative bacteria. As it is essential for the survival of many pathogens, LpxC is an attractive target for antibacterial therapeutics. Herein, we report the product-bound co-crystal structure of LpxC from the acheal Aquifex aeolicus solved to 1.

Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain.

Overexpression of Pseudomonas aeruginosa LpxC with its inhibitors in an acrB-deficient Escherichia coli strain.

In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy.

Synergy of streptogramin antibiotics occurs independently of their effects on translation.

Streptogramin antibiotics are divided into types A and B, which in combination can act synergistically. We compared the molecular interactions of the streptogramin combinations Synercid (type A, dalfopristin; type B, quinupristin) and NXL 103 (type A, flopristin; type B, linopristin) with the Escherichia coli 70S ribosome by X-ray crystallography. We further analyzed the activity of the streptogramin components individually and in combination.

Antibacterial inhibitors of Gram-positive thymidylate kinase: structure-activity relationships and chiral preference of a new hydrophobic binding region.

Thymidylate kinase (TMK), an essential enzyme in bacterial DNA biosynthesis, is an attractive therapeutic target for the development of novel antibacterial agents, and we continue to explore TMK inhibitors with improved potency, protein binding, and pharmacokinetic potential. A structure-guided design approach was employed to exploit a previously unexplored region in Staphylococcus aureus TMK via novel interactions. These efforts produced compound 39, with 3 nM IC50 against S.

Caged mono- and divalent ligands for light-assisted disruption of PDZ domain-mediated interactions.

We report a general method for light-assisted control of interactions of PDZ domain binding motifs with their cognate domains by the incorporation of a photolabile caging group onto the essential C-terminal carboxylate binding determinant of the motif. The strategy was implemented and validated for both simple monovalent and biomimetic divalent ligands, which have recently been established as powerful tools for acute perturbation of native PDZ domain-dependent interactions in live cells.

Sulfonylpiperidines as novel, antibacterial inhibitors of Gram-positive thymidylate kinase (TMK).

Thymidylate kinase (TMK) is an essential enzyme for DNA synthesis in bacteria, phosphorylating deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP), and thus is a potential new antibacterial drug target. Previously, we have described the first potent and selective inhibitors of Gram-positive TMK, leading to in vivo validation of the target. Here, a structure-guided design approach based on the initial series led to the discovery of novel sulfonylpiperidine inhibitors of TMK.

Discovery of selective and potent inhibitors of gram-positive bacterial thymidylate kinase (TMK).

Thymidylate kinase (TMK) is an essential enzyme in bacterial DNA synthesis. The deoxythymidine monophosphate (dTMP) substrate binding pocket was targeted in a rational-design, structure-supported effort, yielding a unique series of antibacterial agents showing a novel, induced-fit binding mode. Lead optimization, aided by X-ray crystallography, led to picomolar inhibitors of both Streptococcus pneumoniae and Staphylococcus aureus TMK.

In vivo validation of thymidylate kinase (TMK) with a rationally designed, selective antibacterial compound.

There is an urgent need for new antibacterials that pinpoint novel targets and thereby avoid existing resistance mechanisms. We have created novel synthetic antibacterials through structure-based drug design that specifically target bacterial thymidylate kinase (TMK), a nucleotide kinase essential in the DNA synthesis pathway. A high-resolution structure shows compound TK-666 binding partly in the thymidine monophosphate substrate site, but also forming new induced-fit interactions that give picomolar affinity.

Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization.

The interactions of the AMPA receptor (AMPAR) auxiliary subunit Stargazin with PDZ domain-containing scaffold proteins such as PSD-95 are critical for the synaptic stabilization of AMPARs. To investigate these interactions, we have developed biomimetic competing ligands that are assembled from two Stargazin-derived PSD-95/DLG/ZO-1 (PDZ) domain-binding motifs using 'click' chemistry. Characterization of the ligands in vitro and in a cellular FRET-based model revealed an enhanced affinity for the multiple PDZ domains of PSD-95 compared to monovalent peptides.

Structural analysis of WbpE from Pseudomonas aeruginosa PAO1: a nucleotide sugar aminotransferase involved in O-antigen assembly.

In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P.

Mapping the structure and conformational movements of proteins with transition metal ion FRET.

Visualizing conformational dynamics in proteins has been difficult, and the atomic-scale motions responsible for the behavior of most allosteric proteins are unknown. Here we report that fluorescence resonance energy transfer (FRET) between a small fluorescent dye and a nickel ion bound to a dihistidine motif can be used to monitor small structural rearrangements in proteins. This method provides several key advantages over classical FRET, including the ability to measure the dynamics of close-range interactions, the use of small probes with short linkers, a low orientation dependence, and the ability to add and remove unique tunable acceptors.

Voltage-dependent opening of HCN channels: Facilitation or inhibition by the phytoestrogen, genistein, is determined by the activation status of the cyclic nucleotide gating ring.

Investigation of the mechanistic bases and physiological importance of cAMP regulation of HCN channels has exploited an arginine to glutamate mutation in the nucleotide-binding fold, an approach critically dependent on the mutation selectively lowering the channel's nucleotide affinity. In apparent conflict with this, in intact Xenopus oocytes, HCN and HCN-RE channels exhibit qualitatively and quantitatively distinct responses to the tyrosine kinase inhibitor, genistein -- the estrogenic isoflavonoid strongly depolarizes the activation mid-point of HCN1-R538E, but not HCN1 channels (+9.8 mV + or - 0.

Crystal structure and catalytic mechanism of PglD from Campylobacter jejuni.

The carbohydrate 2, 4-diacetamido-2, 4, 6-trideoxy-alpha-D-glucopyranose (BacAc(2)) is found in a variety of eubacterial pathogens. In Campylobacter jejuni, PglD acetylates the C4 amino group on UDP-2-acetamido-4-amino-2, 4, 6-trideoxy-alpha-D-glucopyranose (UDP-4-amino-sugar) to form UDP-BacAc(2). Sequence analysis predicts PglD to be a member of the left-handed beta helix family of enzymes.

C-terminal movement during gating in cyclic nucleotide-modulated channels.

Activation of cyclic nucleotide-modulated channels such as CNG and HCN channels is promoted by ligand-induced conformational changes in their C-terminal regions. The primary intersubunit interface of these C termini includes two salt bridges per subunit, formed between three residues (one positively charged and two negatively charged amino acids) that we term the SB triad. We previously hypothesized that the SB triad is formed in the closed channel and breaks when the channel opens.

In vitro biosynthesis of UDP-N,N'-diacetylbacillosamine by enzymes of the Campylobacter jejuni general protein glycosylation system.

In Campylobacter jejuni 2,4-diacetamido-2,4,6-trideoxy-alpha-d-glucopyranose, termed N,N'-diacetylbacillosamine (Bac2,4diNAc), is the first carbohydrate in the glycoprotein N-linked heptasaccharide. With uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a starting point, two enzymes of the general protein glycosylation (Pgl) pathway in C. jejuni (PglF and PglE) have recently been shown to modify this sugar nucleotide to form UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-d-glycopyranose (UDP-4-amino-sugar) [Schoenhofen, I.

A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization.

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The "defect" in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating.

Structural basis for modulation and agonist specificity of HCN pacemaker channels.

The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration. These nonselective cation channels, underlying the I(f), I(h) and I(q) currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by beta-adrenergic agonists.