Publications by authors named "Cheong Wing Lam"

We successfully developed a fluorescent drug sensor from clinically relevant New Delhi metallo-β-lactamase-1 (NDM-1). The F70 residue was chosen to be replaced with a cysteine for conjugation with thiol-reactive fluorescein-5-maleimide to form fluorescent F70Cf, where "f" refers to fluorescein-5-maleimide. Our proteolytic studies of unlabeled F70C and labeled F70Cf monitored by electrospray ionization-mass spectrometry (ESI-MS) revealed that fluorescein-5-maleimide was specifically linked to C70 in 1:1 mole ratio (F70C:fluorophore).

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Rationally designed mutations on recombinant arginine deiminase (ADI) could act as a 'turn-off' L-arginine (L-Arg) fluorescent biosensor and provide an alternative method for rapid determination of L-Arg. Double mutations were introduced on the Cys➔Ser and Thr➔Cys of recombinant ADI, rendering a single cysteine present on the protein surface for the site-specific attachment of a fluorophore, fluorescein-5-maleimide. The double mutations on ADI (265C) and its fluorescein-labelled form (265Cf) conserved the catalytic efficiency of wild-type ADI.

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Moenomycin A, the well-known natural product inhibitor of peptidoglycan glycosyltransferase (PGT), is a large amphiphilic molecule of molecular mass of 1583 g/mol and its bioavailablity as a drug is relatively poor. In searching for small-molecule ligands with high inhibition ability targeting the enzyme, we found that the addition of hydrophobic groups to an isatin-based inhibitor of bacterial PGT significantly improves its inhibition against the enzyme, as well as its antibacterial activity. The improvement in enzymatic inhibition can be attributed to a better binding of the small molecule inhibitor to the hydrophobic region of the membrane-bound bacterial cell wall synthesis enzyme and the plasma membrane.

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A copper(II)-based two-dimensional metal-organic framework with nanosheet structure (CuBDC NS) that possesses peroxidase (POx) mimicking activity was prepared. In the presence of hydrogen peroxide, the system catalyses the oxidation of terephthalic acid to a blue-fluorescent product (excitation = 315 nm; emission = 425 nm). Pyrophosphate has a very strong affinity for Cu ion and blocks the POx-mimicking activity of the CuBDC NS.

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Fluorescent labeling of biomacromolecules to 'light up' biological events through non-invasive methods is of great importance, but is still challenging in terms of fluorophore properties and the labeling methods used. Herein, we designed and synthesized a biocompatible and conformation sensitive tetraphenylethene derivative EPB with aggregation induced emission (AIE) properties. By introducing EPB into TEM-1 β-lactamase (TEM-1 Bla) through a two-step approach, a conformation-dependent fluorescent sensor EPB104-Bla was genetically engineered, which was applied to monitor the protein-protein interaction (PPI) with β-lactamase inhibitor protein (BLIP).

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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→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue 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.

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Herein is described the development of a novel switch-on fluorescence assay for detecting β-lactamases. The fluorescence assay comprises two components: solid beads coated with a β-lactam antibiotic, which is linked to an environment-sensitive fluorophore (dansylaminothiophenol, DTA), and amyloid fibrils of hen lysozyme (acting as fluorescence enhancer and visual tool). In the presence of the clinically significant TEM-1 β-lactamase, the DTA-antibiotic complex on the solid beads is hydrolyzed, thus releasing the DTA dye into solution.

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