One-pot synthesis of indenobenzofurans via tandem Michael addition-elimination and palladium-catalysed C-H activation

A tandem, one-pot reaction of 2,3-dibromoindenone with phenols is reported, which provides a straightforward approach to accomplish structurally diverse indenobenzofurans in excellent yields. Besides, the control experiments revealed that reaction proceeds through a two-step sequence via an Intermolecular Michael addition-elimination reaction, which enables the C-O bond formation, followed by palladium-catalyzed intramolecular C-C bond formation through C-H activation. Significantly, this protocol offers a convenient approach to synthesize the diverse tetracyclic indenobenzofurans

Glycerol Monolaurate and Dodecylglycerol Effects on Staphylococcus aureus and Toxic Shock Syndrome Toxin-1 In Vitro and In Vivo

BACKGROUND:Glycerol monolaurate (GML), a 12 carbon fatty acid monoester, inhibits Staphylococcus aureus growth and exotoxin production, but is degraded by S. aureus lipase. Therefore, dodecylglycerol (DDG), a 12 carbon fatty acid monoether, was compared in vitro and in vivo to GML for its effects on S. aureus growth, exotoxin production, and stability. METHODOLOGY/PRINCIPAL FINDINGS:Antimicrobial effects of GML and DDG (0 to 500 microg/ml) on 54 clinical isolates of S. aureus, including pulsed-field gel electrophoresis (PFGE) types USA200, USA300, and USA400, were determined in vitro. A rabbit Wiffle ball infection model assessed GML and DDG (1 mg/ml instilled into the Wiffle ball every other day) effects on S. aureus (MN8) growth (inoculum 3x10(8) CFU/ml), toxic shock syndrome toxin-1 (TSST-1) production, tumor necrosis factor-alpha (TNF-alpha) concentrations and mortality over 7 days. DDG (50 and 100 microg/ml) inhibited S. aureus growth in vitro more effectively than GML (p<0.01) and was stable to lipase degradation. Unlike GML, DDG inhibition of TSST-1 was dependent on S. aureus growth. GML-treated (4 of 5; 80%) and DDG-treated rabbits (2 of 5; 40%) survived after 7 days. Control rabbits (5 of 5; 100%) succumbed by day 4. GML suppressed TNF-alpha at the infection site on day 7; however, DDG did not (<10 ng/ml versus 80 ng/ml, respectively). CONCLUSIONS/SIGNIFICANCE:These data suggest that DDG was stable to S. aureus lipase and inhibited S. aureus growth at lower concentrations than GML in vitro. However, in vivo GML was more effective than DDG by reducing mortality, and suppressing TNF-alpha, S. aureus growth and exotoxin production, which may reduce toxic shock syndrome. GML is proposed as a more effective anti-staphylococcal topical anti-infective candidate than DDG, despite its potential degradation by S. aureus lipase

IP Policy Forum: Repurposing & Collaborative Drug Development for Rare Diseases

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Conserved Conformational Hierarchy across Functionally Divergent Glycosyltransferases of the GT-B Structural Superfamily as Determined from Microsecond Molecular Dynamics

It has long been understood that some proteins undergo conformational transitions en route to the Michaelis Complex to allow chemistry. Examination of crystal structures of glycosyltransferase enzymes in the GT-B structural class reveals that the presence of ligand in the active site triggers an open-to-closed conformation transition, necessary for their catalytic functions. Herein, we describe microsecond molecular dynamics simulations of two distantly related glycosyltransferases that are part of the GT-B structural superfamily, HepI and GtfA. Simulations were performed using the open and closed conformations of these unbound proteins, respectively, and we sought to identify the major dynamical modes and communication networks that interconnect the open and closed structures. We provide the first reported evidence within the scope of our simulation parameters that the interconversion between open and closed conformations is a hierarchical multistep process which can be a conserved feature of enzymes of the same structural superfamily. Each of these motions involves of a collection of smaller molecular reorientations distributed across both domains, highlighting the complexities of protein dynamic involved in the interconversion process. Additionally, dynamic cross-correlation analysis was employed to explore the potential effect of distal residues on the catalytic efficiency of HepI. Multiple distal nonionizable residues of the C-terminal domain exhibit motions anticorrelated to positively charged residues in the active site in the N-terminal domain involved in substrate binding. Mutations of these residues resulted in a reduction in negatively correlated motions and an altered enzymatic efficiency that is dominated by lower Km values with kcat effectively unchanged. The findings suggest that residues with opposing conformational motions involved in the opening and closing of the bidomain HepI protein can allosterically alter the population and conformation of the “closed” state, essential to the formation of the Michaelis complex. The stabilization effects of these mutations likely equally influence the energetics of both the ground state and the transition state of the catalytic reaction, leading to the unaltered kcat. Our study provides new insights into the role of conformational dynamics in glycosyltransferase’s function and new modality to modulate enzymatic efficiency

Stability of the compounds to <i>Staphylococcus aureus</i> (MN8) lipase.

<p>(A) Glycerol monolaurate (GML). (B) Dodecylglycerol (DDG). Clear zone indicates that the compound was degraded. Arrow denotes of the radius of the clear zone on the slide.</p

Cytotoxicity of GML and DDG to Human Vaginal Epithelial Cells (HVECs).

<p>HVECs were exposed to GML (▪) and DDG (▴) for 6 h. Cytotoxicity was accessed by measuring the release of LDH. Error bars are SEM. The dashed line indicates median cell survival (LD₅₀). Symbols:▪, GML; ▴, DDG.</p

Effects of GML and DDG on <i>Staphylococcus aureus</i> Toxic Shock Syndrome Toxin-1 (TSST-1) production.

<p>(A) <i>S. aureus</i> MN8 was exposed to GML 0, 25 and 50 µg/ml for 6 and 24 h, and bacterial densities at 6 and 24 h were determined by plate counts. (B) The corresponding concentrations of TSST-1 of the above GML experiment. (C) <i>S. aureus</i> MN8 was exposed to DDG 0, 5, 15, and 25 µg/ml for 6 and 24 h. (D) The corresponding concentrations of TSST-1 from above DDG experiments. TSST-1 concentrations are presented as percent of the TSST-1 concentrations in 24 h control samples. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007499#s2" target="_blank">Results</a> are mean±SEM. The dashed line indicates the starting inocula. *, p<0.05.</p

Glycerol monolaurate (GML) inhibition of <i>Staphylococcus aureus</i>.

<p>GML concentrations 50, 100, and 500 µg/ml were tested versus <i>S. aureus</i> isolates from different PFGE types, USA400 MRSA (A), USA400 MSSA (B), USA300 MRSA (C), USA200 MRSA (D), USA200 MSSA (E), atopic dermatitis strains (F), vaginal strains from healthy women (G), for 18 h at 37°C with shaking. The dashed line indicates the starting inocula. Each square (▪) indicates one isolate. The bars represent the mean±SEM of bacterial density in the group.</p

Antistaphylococcal effects of GML and DDG in a rabbit Wiffle ball infection model.

<p>Rabbits (n = 5 in each group) were infected with 3×10⁸ CFU/ml <i>S. aureus</i> MN8, and compounds (final concentration 1 mg/ml) were instilled into the Wiffle balls every-other-day and rabbits monitored up to 7 days. Survival of the rabbits (A), bacterial counts (B), TSST-1 production (C), and TNF-α levels (D) in the Wiffle balls. TSST-1 presented as percent of day 2 TSST-1 concentrations of the control rabbits (GML, close bars; DDG, open bars). Error bars are SEM. Symbols: ○, control; ▪, GML; ▴, DDG; *, p<0.05.</p

Dodecylglycerol (DDG) inhibition of <i>Staphylococcus aureus</i>.

<p>DDG concentrations 25, 50, and 100 µg/ml were tested versus <i>S. aureus</i> isolates from different PFGE types, USA400 MRSA (A), USA400 MSSA (B), USA300 MRSA (C), USA200 MRSA (D), USA200 MSSA (E), atopic dermatitis strains (F), vaginal strains from healthy women (G), for 18 h at 37°C with shaking. The dashed line indicates the starting inocula. Each triangle (▴) indicates one isolate. The bars represent the mean±SEM of bacterial density in the group.</p