Sensitivity improvement in 19F NMR-based screening experiments:: Theoretical considerations and experimental applications
Sensitivity improvement in 19F NMR-based screening experiments:: Theoretical considerations and experimental applications
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DOI：
10.1021/ja0542385
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发表时间：
2005-09-28
影响因子：
15
通讯作者：
Kümmerle, R
中科院分区：
文献类型：
--
作者：
Dalvit, C;Mongelli, N;Kümmerle, R
利用FAXS（氟化学位移各向异性和交换筛选）和3-FABS（用于生化筛选的三个氟原子）进行的基于NMR的结合和功能筛选代表了用于鉴定新型潜在候选药物的高通量筛选的潜在替代方法。该方法目前的主要局限性是其固有的低灵敏度，限制了测试化合物的数量。克服这个问题的一种方法是使用低温冷却的F-19探头，以减少接收器电路中的热噪声。在两种筛选技术实现了新的低温F-19探针技术的灵敏度提高允许增加的吞吐量，检测较弱的粘合剂和抑制剂（相关的片段为基础的铅发现程序），检测慢粘合剂，并减少蛋白质和底物的消耗。这些方面进行了分析与理论模拟和实验定量性能评价。应用3-FABS结合低温F-19探针技术快速筛选在非常低的酶浓度和目前的检测极限达到这种方法也被提出。
NMR-based binding and functional screening performed with FAXS (fluorine chemical shift anisotropy and exchange for screening) and 3-FABS (three fluorine atoms for biochemical screening) represents a potential alternative approach to high-throughput screening for the identification of novel potential drug candidates. The major limitation of this method in its current status is its intrinsic low sensitivity that limits the number of tested compounds. One approach for overcoming this problem is the use of a cryogenically cooled F-19 probe that reduces the thermal noise in the receiver circuitry. Sensitivity improvement in the two screening techniques achieved with the novel cryogenic F-19 probe technology permits an increased throughput, detection of weaker binders and inhibitors (relevant in a fragment-based lead discovery program), detection of slow binders, and reduction in protein and substrate consumption. These aspects are analyzed with theoretical simulations and experimental quantitative performance evaluation. Application of 3-FABS combined with the cryogenic F-19 probe technology to rapid screening at very low enzyme concentrations and the current detection limits reached with this approach are also presented.