18 research outputs found
Chemical Perturbation of Secondary Metabolism Demonstrates Important Links to Primary Metabolism
SummaryBacterially produced secondary metabolites are used as antibiotics, anticancer drugs, and for many other medicinal applications. The mechanisms that limit the production of these molecules in the laboratory are not well understood, and this has impeded the discovery of many important compounds. We have identified small molecules that remodel the yields of secondary metabolites in many actinomycetes and show that one set of theseĀ molecules does so by inhibiting fatty acid biosynthesis. This demonstrates a particularly intimate relationship between this primary metabolic pathway and secondary metabolism and suggests an approach to enhance the yields of metabolites for discovery and biochemical characterization
Carbon Monoxide-Releasing Molecule-401 Suppresses Polymorphonuclear Leukocyte Migratory Potential by Modulating F-Actin Dynamics
Carbon monoxide-releasing molecules (CORMs) suppress inflammation by reducing polymorphonuclear leukocyte (PMN) recruitment to the affected organs. We investigated modulation of PMN-endothelial cell adhesive interactions by water-soluble CORM-401 using an experimental model of endotoxemia in vitro. Human umbilical vein endothelial cells (HUVEC) grown on laminar-flow perfusion channels were stimulated with 1 Ī¼g/mL lipopolysaccharide for 6 hours and perfused with 100 Ī¼mol/L CORM-401 (or inactive compound iCORM-401)-pretreated PMN for 5 minutes in the presence of 1.0 dyn/cm
HUVEC: PMN co-cultures were perfused for additional 15 minutes with PMN-free medium containing CORM-401/inactive CORM-401. The experiments were videorecorded (phase-contrast microscopy), and PMN adhesion/migration were assessed off-line. In parallel, CORM-401-dependent modulation of PMN chemotaxis, F-actin expression/distribution, and actin-regulating pathways [eg, p21-activated protein kinases (PAK1/2) and extracellular signal-regulated kinase (ERK)/C-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK)] were assessed in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Pretreating PMN with CORM-401 did not suppress PMN adhesion to HUVEC, but significantly reduced PMN transendothelial migration (P \u3c 0.0001) and fMLP-induced PMN chemotaxis (ie, migration directionality and velocity). These changes were associated with CORM-401-dependent suppression of F-actin levels/cellular distribution and fMLP-induced phosphorylation of PAK1/2 and ERK/JNK MAPK (P \u3c 0.05). CORM-401 had no effect on p38 MAPK activation. In summary, this study demonstrates, for the first time, CORM-401-dependent suppression of neutrophil migratory potential associated with modulation of PAK1/2 and ERK/JNK MAPK signaling and F-actin dynamics
A sputum bioassay for airway eosinophilia using an eosinophil peroxidase aptamer
Abstract Eosinophils are granulocytes that play a significant role in the pathogenesis of asthma and other airway diseases. Directing patient treatment based on the level of eosinophilia has been shown to be extremely effective in reducing exacerbations and therefore has tremendous potential as a routine clinical test. Herein, we describe the in vitro selection and optimization of DNA aptamers that bind to eosinophil peroxidase (EPX), a protein biomarker unique to eosinophils. Fifteen rounds of magnetic bead aptamer selection were performed prior to high throughput DNA sequencing. The top 10 aptamer candidates were assessed for EPX binding using a mobility shift assay. This process identified a lead aptamer candidate termed EAP1-05 with low nanomolar affinity and high specificity for EPX over other common sputum proteins. This aptamer sequence was further optimized through truncation and used to develop an easy-to-use colourimetric pull-down assay that can detect EPX over a concentration range from 1 ā 100Ā nM in processed sputum. Forty-six clinical samples were processed using a new sputum dispersal method, appropriate for a rapid assessment assay, that avoids centrifugation and lengthy processing times. The assay showed 89% sensitivity and 96% specificity to detect eosinophilia (compared to gold standard sputum cytometry), with results being produced in under an hour. This assay could allow for an easy assessment of eosinophil activity in the airway to guide anti-inflammatory therapy for several airway diseases
Transition State Analysis of Acid-Catalyzed Hydrolysis of an Enol Ether, Enolpyruvylshikimate 3āPhosphate (EPSP)
Proton transfer to carbon represents a significant catalytic
challenge
because of the large intrinsic energetic barrier and the frequently
unfavorable thermodynamics. Multiple kinetic isotope effects (KIEs)
were measured for acid-catalyzed hydrolysis of the enol ether functionality
of enolpyruvylshikimate 3-phosphate (EPSP) as a nonenzymatic analog
of the EPSP synthase (AroA) reaction. The large solvent deuterium
KIE demonstrated that protonating C3 was the rate-limiting step, and
the lack of solvent hydron exchange into EPSP demonstrated that protonation
was irreversible. The reaction mechanism was stepwise, with C3, the
methylene carbon, being protonated to form a discrete oxacarbenium
ion intermediate before water attack at the cationic center, that
is, an A<sub>H</sub><sup>ā”</sup>*A<sub>N</sub> (or A<sub>H</sub><sup>ā”</sup> + A<sub>N</sub>) mechanism. The calculated 3-<sup>14</sup>C and 3,3-<sup>2</sup>H<sub>2</sub> KIEs varied as a function
of the extent of proton transfer at the transition state, as reflected
in the C3āH<sup>+</sup> bond order, <i>n</i><sub>C3āH+</sub>. The calculated 3-<sup>14</sup>C KIE was a function
primarily of C3 coupling with the movement of the transferring proton,
as reflected in the reaction coordinate contribution (<sup>light</sup>Ī½<sup>ā”</sup>/<sup>heavy</sup>Ī½<sup>ā”</sup>), rather than of changes in bonding. Coupling was strongest in early
and late transition states, where the reaction coordinate frequency
was lower. The other calculated <sup>14</sup>C and <sup>18</sup>O
KIEs were more sensitive to interactions with counterions and solvation
in the model structures than <i>n</i><sub>C3āH+</sub>. The KIEs revealed a moderately late transition state with significant
oxacarbenium ion character and with a C3āH<sup>+</sup> bond
order ā0.6
Fluorous Analogue of Chloramine-T: Preparation, Xāray Structure Determination, and Use as an Oxidant for Radioiodination and sāTetrazine Synthesis
A fluorous oxidant that can be used
to introduce radioiodine into
small molecules and proteins and generate iodinated tetrazines for
bioorthogonal chemistry has been developed. The oxidant was prepared
in 87% overall yield by combining a fluorous amine with tosyl chloride,
followed by chlorination using aqueous sodium hypochlorite. A crystal
structure of the oxidant, which is a fluorous analogue of chloramine-T,
was obtained. The compound was shown to be stable for 7 days in EtOH
and for longer than three months as a solid. The oxidant was effective
at promoting the labeling of arylstannanes using [<sup>125</sup>I]ĀNaI,
where products were isolated in high specific activity in yields ranging
from 46% to 86%. Similarly, iodinated biologically active proteins
(e.g., thrombin) were successfully produced, as well as a radioiodinated
tetrazine, through a concomitant oxidation-halodemetalation reaction.
Because of its fluorous nature, unreacted oxidant and associated reaction
byproducts can be removed quantitatively from reaction mixtures by
passing solutions through fluorous solid phase extraction cartridges.
This feature enables rapid and facile purification, which is critical
when working with radionuclides and is similarly beneficial for general
synthetic applications