School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China.
Pharmaceutical and Medicinal Chemistry (Computer-Aided Drug Design), Institute of Pharmacy, Freie Universität Berlin, Berlin, Germany.
Mol Vis. 2021 Oct 6;27:601-607. eCollection 2021.
Polymorphisms in the gene that codes for the human cytochrome P450 enzyme CYP4V2 are a cause of Bietti crystalline dystrophy (BCD). Therefore, inhibition of CYP4V2 activity may well be a cause of visual disability. However, monitoring the fatty acid hydroxylation reactions catalyzed by this enzyme is tedious and not well suited for inhibitor screening.
We investigated the use of proluciferin compounds as probe substrates for efficient and convenient determination of CYP4V2 activity.
Ten proluciferins were tested for conversion by CYP4V2, and eight were found to be substrates of this enzyme. One point inhibitor assays were performed using luciferin 6' 3-furfuryl ether methyl ester (luciferin-3FEME) as the probe substrate and 12 test compounds. As expected, HET0016 had by far the strongest effect, while two other compounds (including osilodrostat) also displayed statistically significant inhibitory potency. The half maximal inhibitory concentration (IC) for HET0016 was determined to be 179 nM. A recently identified potent inhibitor of human CYP4Z1 was found not to inhibit CYP4V2. To explore the selectivity of this compound between CYP4Z1 and CYP4V2, we developed a homology model of CYP4V2 and conducted docking experiments.
We provide the first protocol for a robust and convenient CYP4V2 inhibitor assay that does not depend on fatty acid analysis but can be simply monitored with luminescence. Moreover, we demonstrate additional evidence for the concern that compounds with CYP-inhibitory properties may inhibit CYP4V2 activity and thus, possibly cause visual disability.
编码人细胞色素 P450 酶 CYP4V2 的基因中的多态性是 Bietti 结晶状营养不良（BCD）的一个原因。因此，CYP4V2 活性的抑制很可能是视力障碍的一个原因。然而，监测该酶催化的脂肪酸羟化反应既繁琐又不适合抑制剂筛选。
我们研究了使用原荧光素化合物作为探针底物来有效和方便地测定 CYP4V2 活性的方法。
测试了 10 种原荧光素化合物被 CYP4V2 转化的情况，其中 8 种被发现是该酶的底物。使用荧光素 6' 3-呋喃基醚甲酯（luciferin-3FEME）作为探针底物和 12 种测试化合物进行了一点抑制剂测定。正如预期的那样，HET0016 的效果迄今为止最强，而另外两种化合物（包括 osilodrostat）也显示出具有统计学意义的抑制效力。HET0016 的半最大抑制浓度（IC）被确定为 179 nM。最近发现的一种有效的人类 CYP4Z1 抑制剂被发现不抑制 CYP4V2。为了探索该化合物在 CYP4Z1 和 CYP4V2 之间的选择性，我们开发了 CYP4V2 的同源模型并进行了对接实验。
我们提供了第一个稳健、方便的 CYP4V2 抑制剂测定方案，该方案不依赖于脂肪酸分析，而可以通过发光简单地进行监测。此外，我们提供了更多的证据表明，具有 CYP 抑制特性的化合物可能抑制 CYP4V2 活性，从而可能导致视力障碍。
