کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10584170 | 981326 | 2013 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
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
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موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آلی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
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 ZⲠfactor of 0.8. The automated assay is an epi-absorbance endpoint assay in 384-well plates, has a ZⲠfactor 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Bioorganic & Medicinal Chemistry - Volume 21, Issue 11, 1 June 2013, Pages 3138-3146
Journal: Bioorganic & Medicinal Chemistry - Volume 21, Issue 11, 1 June 2013, Pages 3138-3146
نویسندگان
Pei W. Thomas, Timothy Spicer, Michael Cammarata, Jennifer S. Brodbelt, Peter Hodder, Walter Fast,