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.
Bioorg Med Chem
; 21(11): 3138-46, 2013 Jun 01.
Article
en En
| MEDLINE
| ID: mdl-23591260
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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Zinc
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Nitroprusiato
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Ácido p-Cloromercuribenzoico
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Inhibidores de beta-Lactamasas
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Antibacterianos
Tipo de estudio:
Diagnostic_studies
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Prognostic_studies
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Screening_studies
Idioma:
En
Revista:
Bioorg Med Chem
Asunto de la revista:
BIOQUIMICA
/
QUIMICA
Año:
2013
Tipo del documento:
Article
País de afiliación:
Estados Unidos