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.
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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DOI：
10.1016/j.bmc.2013.03.031
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发表时间：
2013-06-01
影响因子：
3.5
通讯作者：
Fast, Walter
中科院分区：
文献类型：
--
作者：
Thomas, Pei W.;Spicer, Timothy;Cammarata, Michael;Brodbelt, Jennifer S.;Hodder, Peter;Fast, Walter
由于新德里金属-β-内酰胺酶-1（NDM-1）介导的抗生素耐药性的全球威胁以及缺乏针对该酶的结构多样性抑制剂，我们开发了手动和自动格式的筛选和反筛选测定。手动测定是96孔板中基于跨孔吸光度的终点测定，Z'因子为0.8。自动化测定是384孔板中的落射吸光度终点测定，具有≥ 0.8的Z'因子，良好的信号/基线比（> 3.8），并且可能可扩展用于高通量筛选（HTS）。还提出了基于TEM-1的计数器筛选，以消除由于测定干扰或脱靶活性而导致的假阳性。经初步筛选的化合物库鉴定出两种巯基修饰化合物为真正的NDM-1抑制剂：对氯香豆素苯甲酸酯（p-CMB）和硝普钠。重组NDM-1具有一个Cys残基，其用作保守的活性位点主要锌配体，并且如通过LC-MS/MS确认的那样被p-CMB选择性修饰。然而，C208 D突变导致酶保持几乎完全的内酰胺酶活性，但对抑制剂完全耐药。这些结果预测共价靶向保守的活性位点Cys残基作为药物设计策略可能具有缺点。
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-hloromecuribenzoate (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.
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影响因子：
7.3
作者：
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通讯作者：
Shoichet, Brian K.
影响因子：
3.5
作者：
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