Zhang Xiang, He Shu, Hu Xiaolei, Wu Jing, Li Xinpeng, Liao Fei, Yang Xiaolan
Key Laboratory of Clinical Laboratory Diagnosis of the Education Ministry, College of Laboratory Medicine, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing 400016, China.
School of Pharmacy and Bioengineering, Chongqing University of Technology, Lijiatuo, Chongqing 400054, China.
Comb Chem High Throughput Screen. 2019;22(1):49-58. doi: 10.2174/1386207322666190306142810.
Human full-length cyclic nucleotide phosphodiesterase isozyme 4B2 (hPDE4B2) as the target for screening and characterizing inhibitors suffers from low activity yield and the coexistence of two conformational states bearing different affinities for (R)-rolipram. Hence, the 152~528 truncate of hPDE4B2 existing only in the low-affinity conformation state for (R)-rolipram was compared against the full-length hPDE4B2 to characterize inhibitors.
With 6His-SUMO tag at the N-terminus, both the full-length hPDE 4B2 (SF-hPDE4B2) and the 152~528 truncate (ST-hPDE4B2) were expressed in Escherichia coli cells, purified through Ni-NTA column and compared for the characterization of inhibitors. The inhibition constants (Ki) of some synthesized rolipram analogues against both targets were determined with 96-well microplate through the coupled action of monophosphatase on AMP and spectrophotometric assay of phosphate with malachite green.
After affinity purification with Ni2+-NTA column, ST-hPDE4B2 showed about 30-fold higher specific activity and 100-fold higher activity yield than SF-hPDE4B2; Ki of (R)-rolipram on ST-hPDE4B2 was consistent with that on the low-affinity state of the untagged full-length hPDE4B2 expressed in insect cells. Of some representative rolipram analogues as inhibitors, a dual-logarithm model quantitatively described their monotonic association, and Ki from 0.010 mM to 8.5 mM against SF-hPDE4B2 was predicted from Ki against ST-hPDE4B2, supporting the discovery of consistent hits by the use of both targets with a pair of properly-set cutoffs.
ST-hPDE4B2 with much higher activity yield may be a favorable alternative target to characterize/screen rolipram analogues as hPDE4B inhibitors in high-throughput mode.
人全长环核苷酸磷酸二酯酶同工酶4B2（hPDE4B2）作为筛选和鉴定抑制剂的靶点，存在活性产率低以及两种对（R）-咯利普兰具有不同亲和力的构象状态共存的问题。因此，将仅以低亲和力构象状态存在的hPDE4B2的152~528截短体与全长hPDE4B2进行比较，以鉴定抑制剂。
在N端带有6His-SUMO标签的全长hPDE 4B2（SF-hPDE4B2）和152~528截短体（ST-hPDE4B2）均在大肠杆菌细胞中表达，通过镍-亚氨基二乙酸（Ni-NTA）柱纯化，并比较用于抑制剂的鉴定。通过单磷酸酶对AMP的偶联作用以及用孔雀石绿对磷酸盐进行分光光度测定，用96孔微孔板测定了一些合成的咯利普兰类似物对两个靶点的抑制常数（Ki）。
用Ni2+-NTA柱进行亲和纯化后，ST-hPDE4B2的比活性比SF-hPDE4B2高约30倍，活性产率高100倍；（R）-咯利普兰对ST-hPDE4B2的Ki与在昆虫细胞中表达的无标签全长hPDE4B2的低亲和力状态下的Ki一致。对于一些作为抑制剂的代表性咯利普兰类似物，双对数模型定量描述了它们的单调结合，并且根据对ST-hPDE4B2的Ki预测了对SF-hPDE4B2的Ki为0.010 mM至8.5 mM，支持通过使用两个靶点并设置一对适当的截止值来发现一致的命中物。
具有更高活性产率的ST-hPDE4B2可能是在高通量模式下作为hPDE4B抑制剂鉴定/筛选咯利普兰类似物的有利替代靶点。
