title: An altered zinc-binding site confers resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by high-throughput screening.
abstract: Due to the global threat of antibiotic resistance mediated by New Delhi metallo-beta-lactamase-1 (NDM-1) and the lack of structurally diverse inhibitors reported for this enzyme, we developed screening and counter-screening assays for manual and automated formats. The manual assay is a trans-well absorbance-based endpoint assay in 96-well plates and has a Z' factor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a Z' factor of ≥0.8, good signal/baseline ratios (>3.8), and is likely scalable for high-throughput screening (HTS). A TEM-1-based counter-screen is also presented to eliminate false positives due to assay interference or off-target activities. A pilot screen of a pharmacologically characterized compound library identified two thiol-modifying compounds as authentic NDM-1 inhibitors: p-chloromecuribenzoate (p-CMB) and nitroprusside. Recombinant NDM-1 has one Cys residue that serves as a conserved active-site primary zinc ligand and is selectively modified by p-CMB as confirmed by LC-MS/MS. However a C208D mutation results in an enzyme that maintains almost full lactamase activity, yet is completely resistant to the inhibitor. These results predict that covalent targeting of the conserved active-site Cys residue may have drawbacks as a drug design strategy.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, TX 78712, USA.
authors.forename: Pei W
authors.initials: PW
authors.lastname: Thomas
authors.forename: Timothy
authors.initials: T
authors.lastname: Spicer
authors.forename: Michael
authors.initials: M
authors.lastname: Cammarata
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
authors.forename: Walter
authors.initials: W
authors.lastname: Fast
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry*; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004755:Enterobacteriaceae / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D057166:High-Throughput Screening Assays; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009599:Nitroprusside / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D053719:Tandem Mass Spectrometry; D015032:Zinc / Q000737:chemistry*; D018440:beta-Lactam Resistance; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics; D020245:p-Chloromercuribenzoic Acid / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D065093:beta-Lactamase Inhibitors; D009599:Nitroprusside; D020245:p-Chloromercuribenzoic Acid; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1; D015032:Zinc; D003545:Cysteine