Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening

Wing Lam Cheong, Ming San Tsang, Pui Kin So, Wai Hong Chung, Yun Chung Leung, Pak Ho Chan

Research output: Journal article publicationJournal articleAcademic researchpeer-review

9 Citations (Scopus)

Abstract

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216mutation and fluorophore labelling for in vitro drug screening. The Val216residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening.
Original languageEnglish
Article numbere00136
Pages (from-to)523-533
Number of pages11
JournalBioscience Reports
Volume34
Issue number5
DOIs
Publication statusPublished - 1 Jan 2014

Keywords

  • Antibiotics
  • Bacteria
  • Drug screening
  • Inhibitors sensor
  • β-lactamase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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