There
is currently no ideal radiotracer for imaging of protein
synthesis rate (PSR) by positron emission tomography (PET). Existing
fluorine-18-labeled amino acid-based radiotracers predominantly visualize
amino acid transporter processes, and in many cases they are not incorporated
into nascent proteins at all. Others are radiolabeled with the short-half-life
positron emitter carbon-11, which is rather impractical for many PET
centers. Based on the puromycin (6) structural manifold,
a series of 10 novel derivatives of 6 was prepared via
Williamson ether synthesis from a common intermediate. A bioluminescence
assay was employed to study their inhibitory action on protein synthesis,
which identified the fluoroethyl analogue 7b as a lead
compound. The fluorine-18 analogue was prepared via nucleophilic substitution
of the corresponding tosylate precursor in a modest radiochemical
yield of 2 ± 0.6% with excellent radiochemical purity (>99%)
and showed complete stability over 3 h at ambient temperature.
Gaussia luciferase (GLuc) is a secreted protein with significant potential for use as a reporter of gene expression in bacterial pathogenicity studies. To date there are relatively few examples of its use in bacteriology. In this study we show that GLuc can be functionally expressed in the human pathogen Staphylococcus aureus and furthermore show that it can be used as a biosensor for the agr quorum sensing (QS) system which employs autoinducing peptides to control virulence. GLuc was linked to the P3 promoter of the S. aureusagr operon. Biosensor strains were validated by evaluation of chemical agent-mediated activation and inhibition of agr. Use of GLuc enabled quantitative assessment of agr activity. This demonstrates the utility of Gaussia luciferase for in vitro monitoring of agr activation and inhibition.
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