<i>In vitro</i> activity of recombinant GH3-6.

<p>(A) The expression of recombinant GH3-6 protein, which was used for the inhibition assays shown in Figs. 2 and 4, was tested by separating 10 µl of His GraviTrap column elution fractions (E1–E3) on a 4–12% polyacrylamide gel followed by Coomassie Brilliant Blue staining (upper panel) or immunodetection using a monoclonal antibody raised against poly-histidine (lower panel). The molecular mass standards (Precision Plus Protein all blue, BioRad) are indicated. The band with the size of approximately 70 kDa corresponds to the His-tagged GH3-6 protein. (B) TLC analysis of GH3-6 enzyme reactions with IAA and 20 amino acids (single letter code). The spot near the origin for the reactions with Trp represents the unbound amino acid. Plates were stained with Ehmann's reagent to detect indole compounds.</p

A New 1,2,3-Triazole Scaffold with Improved Potency against Staphylococcus aureus Biotin Protein Ligase

Staphylococcus aureus, a key ESKAPE bacteria, is responsible for most blood-based infections and, as a result, is a major economic healthcare burden requiring urgent attention. Here, we report in silico docking, synthesis, and assay of N1-diphenylmethyl triazole-based analogues (7–13) designed to interact with the entire binding site of S. aureus biotin protein ligase (SaBPL), an enzyme critical for the regulation of gluconeogenesis and fatty acid biosynthesis. The second aryl ring of these compounds enhances both SaBPL potency and whole cell activity against S. aureus relative to previously reported mono-benzyl triazoles. Analogues 12 and 13, with added substituents to better interact with the adenine binding site, are particularly potent, with Ki values of 6.01 ± 1.01 and 8.43 ± 0.73 nM, respectively. These analogues are the most active triazole-based inhibitors reported to date and, importantly, inhibit the growth of a clinical isolate strain of S. aureus ATCC 49775, with minimum inhibitory concentrations of 1 and 8 μg/mL, respectively

AIEP inhibits GH3-1 and GH3-6 in a competitive manner regarding both substrates – MgATP and IAA.

<p>Reciprocal initial velocities (Fig. 2) for two substrate conentrations were replotted against inhibitor concentrations. (A) Dixon plot for GH3-1 and (B) GH3-6 with varying concentrations of inhibitor and 100 µM (◊) and 1000 µM (○) MgATP and for (C) GH3-1 and (D) GH3-6 with varying concentrations of inhibitor and 10 µM (□) and 100 µM (Δ) IAA. All data represent mean ± standard error of the mean (n = 3).</p

Design of AIEP.

<p>(A) gives the structure of AIEP and (B) shows the structure of the proposed reaction intermediate.</p

Inhibition of GH3-1 and GH3-6 by AIEP.

<p>All kinetic parameters are expressed as means ± standard error of the mean (n = 3).</p

Kinetic analysis of the effect of AIEP on the binding of MgATP and IAA by GH3-1 and GH3-6.

<p>The activity of GH3-1 (A) and GH3-6 (B) was determined over a concentration range of MgATP (1 mM IAA, 1 mM Asp) by quantifying the formation of IAA-Asp after 10 min reaction time using LC-ESI-MS/MS. The same analysis was performed over a concentration range of IAA (3 mM MgATP, 1 mM Asp) using either GH3-1 (C) or GH3-6 (D). The inhibitor concentration in the reaction mix was 0 µM (•), 0.1 µM (○), 1 µM (▴), 5 µM (Δ), 10 µM (▪), or 50 µM (□). The plotted initial velocities were fitted to the Michaelis-Menten equation using non-linear regression (SigmaPlot 11.0). All data represent mean ± standard error of the mean (n = 3).</p

Effect of AIEP on auxin levels and the formation of auxin-Asp conjugates in Shiraz berries.

<p>(A) IAA, (B) IAA-Asp and (C) NAA-Asp were quantified by LC-ESI-MS/MS in <i>ex planta</i> Shiraz berry tissues 5 weeks prior to the initiation of ripening which had been exposed to a Control solution, 0.5 µM NAA (N), 20 µM inhibitor (I(20)) and 0.5 µM NAA in combination with 20 µM (I(20)+N), 10 µM (I(10)+N) or 5 µM (I(5)+N) inhibitor. For each treatment 20 berries were placed on 0.8% agar plates containing the indicated compounds and the plates were kept in the dark at room temperature for 6 h (dark grey bars) or 24 h (light grey bars). FW, fresh weight; n.d., not detected. All data represent mean ± standard error of the mean (n = 3). In each subfigure, bars denoted by a different letter differ significantly (p<0.05) using one-way ANOVA to compare the means followed by Duncan's post hoc test (a–c, 6 h; a′–b′, 24 h).</p

New Series of BPL Inhibitors To Probe the Ribose-Binding Pocket of <i>Staphylococcus aureus</i> Biotin Protein Ligase

Replacing the labile adenosinyl-substituted phosphoanhydride of biotinyl-5′-AMP with a N1-benzyl substituted 1,2,3-triazole gave a new truncated series of inhibitors of <i>Staphylococcus aureus</i> biotin protein ligase (<i>Sa</i>BPL). The benzyl group presents to the ribose-binding pocket of <i>Sa</i>BPL based on <i>in silico</i> docking. Halogenated benzyl derivatives (<b>12t</b>, <b>12u</b>, <b>12w</b>, and <b>12x</b>) proved to be the most potent inhibitors of <i>Sa</i>BPL. These derivatives inhibited the growth of <i>S. aureus</i> ATCC49775 and displayed low cytotoxicity against HepG2 cells

SR2067 Reveals a Unique Kinetic and Structural Signature for PPARγ Partial Agonism

Synthetic full agonists of PPARγ have been prescribed for the treatment of diabetes due to their ability to regulate glucose homeostasis and insulin sensitization. While the use of full agonists of PPARγ has been hampered due to severe side effects, partial agonists have shown promise due to their decreased incidence of such side effects in preclinical models. No kinetic information has been forthcoming in regard to the mechanism of full versus partial agonism of PPARγ to date. Here, we describe the discovery of a partial agonist, SR2067. A co-crystal structure obtained at 2.2 Å resolution demonstrates that interactions with the β-sheet are driven exclusively via hydrophobic interactions mediated through a naphthalene group, an observation that is unique from other partial agonists. Surface plasmon resonance revealed that SR2067 binds to the receptor with higher affinity (<i>K</i>_D = 513 nM) as compared to that of full agonist rosiglitazone, yet it has a much slower off rate compared to that of rosiglitazone

Halogenation of Biotin Protein Ligase Inhibitors Improves Whole Cell Activity against <i>Staphylococcus aureus</i>

We report the synthesis and evaluation of 5-halogenated-1,2,3-triazoles as inhibitors of biotin protein ligase from <i>Staphylococcus aureus</i>. The halogenated compounds exhibit significantly improved antibacterial activity over their nonhalogenated counterparts. Importantly, the 5-fluoro-1,2,3-triazole compound <b>4c</b> displays antibacterial activity against <i>S. aureus</i> ATCC49775 with a minimum inhibitory concentration (MIC) of 8 μg/mL