Comparison of the Full-Length and 152~528 Truncate of Human Cyclic Nucleotide Phosphodiesterase 4B2 for the Characterization of Inhibitors

Aim and Objective: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.