Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe
Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe
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DOI：
10.1006/abio.1999.4348
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发表时间：
1999-12-15
影响因子：
2.9
通讯作者：
Nicoll-Griffith, DA
中科院分区：
文献类型：
--
作者：
Chauret, N;Tremblay, N;Nicoll-Griffith, DA
评价CYP 3A抑制的标准方法是研究在不存在和存在潜在抑制剂的情况下，人肝微粒体中特异性CYP 3A探针睾酮转化为其6 β-羟基代谢产物。通过HPLC实现6 β-羟基代谢物的定量，导致冗长且耗时的测定。为了增加P450抑制通量，努力寻找将产生荧光代谢物的CYP 3A探针。本文报道了DFB作为一种潜在的CYP 3A荧光探针的发现。DFB在人微粒体中显著代谢（类似于1-2 nmol/（min mg蛋白质）），得到荧光化合物DFH。使用多种方法确定了CYP 3A在DFB代谢中的参与。首先，与由表达单一CYP的细胞系（Gentest Supersomes）制备的微粒体进行孵育，表明CYP 3A在DFB代谢中起主要作用。其次，使用CYP 3A抗体进行的免疫抑制研究导致HLM中DFB代谢抑制>95%。第三，特异性CYP 1A 1、1A 2、2C 8/9、2C 19、2D 6和2 E1化学抑制剂的抑制研究未抑制HLM中的DFB活性。然而，酮康唑，咪康唑，尼卡地平，硝苯地平，所有已知的CYP 3A抑制剂，完全消除的DFH的形成在HLM。使用DFB和睾酮作为CYP 3A探针测定的几种抑制剂的效价一致（R = 0.98）。最后，当DFB和睾酮分别与各种人肝微粒体制剂孵育时，DFH的形成和6 β-羟基睾酮的产生获得了良好的一致性（R = 0.94，N = 11）。为了在96孔板中使用DFH作为荧光CYP 3A标记物，重要的是在孵育结束时去除过量的NADPH，因为NADPH的荧光干扰DFH检测。这是通过添加氧化型谷胱甘肽和谷胱甘肽还原酶将NADPH转化为不发荧光的NADP（+）来实现的。液体处理步骤在96孔板格式中完全自动化，并设计模板以生成IC 50曲线并解决供试化合物的潜在荧光干扰。发现该测定具有重现性（日内变异性
The standard method to evaluate CYP3A inhibition is to study the conversion of the specific CYP3A probe testosterone to its 6 beta-hydroxy metabolite in human liver microsomes, in the absence and presence of potential inhibitors. Quantification of the 6 beta-hydroxy metabolite is achieved by HPLC resulting in a tedious and time-consuming assay. In order to increase the P450 inhibition throughput, efforts were made to find a CYP3A probe that would produce a fluorescent metabolite. This paper reports the discovery of DFB as a potential CYP3A fluorescent probe. DFB was significantly metabolized in human microsomes (similar to 1-2 nmol/(min mg protein)) to give the fluorescent compound DFH. The involvement of: CYP3A in the metabolism of DFB was determined using multiple approaches. First, incubations conducted with microsomes made from cell lines expressing single CYPs (Gentest Supersomes) indicated that CYP3A played a major role in the metabolism of DFB. Secondly, immunoinhibition studies conducted with CYP3A antibody resulted in >95% inhibition of DFB metabolism in HLM. Thirdly, inhibition studies with specific CYP1A1, 1A2, 2C8/9, 2C19, 2D6, and 2E1 chemical inhibitors did not suppress DFB activity in HLM. However, ketoconazole, miconazole, nicardipine, and nifedipine, all known CYP3A inhibitors, completely abolished the formation of DFH in HLM. The potency of several inhibitors determined using DFB and testosterone as CYP3A probes was consistent (R = 0.98). Finally, a good agreement was obtained for the formation of DFH and production of 6 beta-hydroxytestosterone when DFB and testosterone were incubated separately with various human liver microsome preparations (R = 0.94, N = 11). In order to use DFH as a fluorescent CYP3A marker in a 96-well plate format, it was important to remove the excess of NADPH at the end of the incubation because the fluorescence of NADPH interferes with DFH detection. This was achieved by adding oxidized glutathione and glutathione reductase to convert NADPH to NADP(+) which is not fluorescent. The liquid-handling steps were fully automated in a 96-well plate format and a template was designed to generate IC50 curves and to address potential fluorescent interferences from the test compounds. The assay was found to be reproducible (intraday variability