A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe
A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe
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使用 [1,3]dioxolo[4,5-f][1,3] 苯并间二氧杂环戊烯 (DBD) 配体探针对铜绿假单胞菌乙酰多胺酰胺水解酶进行基于荧光寿命的结合测定
DOI：
10.1007/s00216-014-7886-5
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发表时间：
2014
影响因子：
4.3
通讯作者：
Meyer-Almes FJ
中科院分区：
文献类型：
--
作者：
Meyners C;Wawrzinek R;Krämer A;Hinz S;Wessig P;Meyer-Almes FJ
用于药物筛选应用的高通量分析必须满足特定的规格。除了能够识别即使是低效价的化合物外，主要问题之一是将筛选活动中的假阳性命中数量降至最低，以减少后续樱桃采摘和确认程序的后勤工作。在这方面，荧光寿命(Flt)似乎是一个理想的读出参数，应该对自动荧光和光吸收化合物具有很强的抵抗力，这是系统假阳性的最常见来源。利用最近发现的[1，3]二氧杂并[4，5-f][1，3]苯并二恶英染料的特殊荧光特性，建立了一种基于荧光光电子显微镜的结合分析方法，具有非常好的读数。该分析体系经过全面验证，不仅符合强大的高通量筛选分析的所有要求，而且适合于确定抑制剂与组蛋白脱乙酰基酶家族酶的准确结合常数。利用所描述的结合试验，鉴定了第一批针对铜绿假单胞菌该酶家族三个成员的抑制剂。从活性和选择性两方面对化合物进行了表征。这种新的配体探针也应该适用于被N-羟基-N‘-苯二胺抑制的组蛋白脱乙酰基酶家族的其他同系物。
High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1, 3] dioxolo [4, 5-f][1, 3] benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N′-phenyloctandiamide.
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原核多胺脱乙酰基酶的结构揭示了与真核组织蛋白脱乙酰基酶的进化功能关系。
影响因子：
2.9
作者：
Lombardi PM;Angell HD;Whittington DA;Flynn EF;Rajashankar KR;Christianson DW
通讯作者：
Christianson DW
基于荧光寿命的 Abelson 激酶测定
影响因子：
--
作者：
S. Pritz;Gabriele Meder;K. Doering;P. Drueckes;Julian Woelcke;L. Mayr;U. Hassiepen
通讯作者：
U. Hassiepen
影响因子：
3.7
作者：
Shen A;Lupardus PJ;Morell M;Ponder EL;Sadaghiani AM;Garcia KC;Bogyo M
通讯作者：
Bogyo M
四参数 Logistic 模型在生物测定中的应用：与斜率和平行线模型的比较。
影响因子：
1.9
作者：
A. Vølund
通讯作者：
A. Vølund
影响因子：
2.9
作者：
Riester, Daniel;Hildmann, Christian;Meyer-Almes, Franz-Josef
通讯作者：
Meyer-Almes, Franz-Josef