| Human cyclic nucleotide phosphodiesterase (PDE) isozymes are important targets for screening inhibitors as potential drugs. Valuable ligands are found via rational design or combination libraries. Classcial high-throughput screening approaches effectively reduce costs and improve efficiency for the discovery of hits of ligands, but require the purity of samples more than 80%. Via the coupled action of a phosphatase on adenosine-5-monophosphate and improved malachite green assay of phosphate, a spectrophotometric assay of PDE activity has been developed; through competitive binding and magnetic recovery of target-ligand complexes, a facile method for the screening of ligands in mixtures has been developed as well. This project studied high-throughput screening of PDE4 inhibitors with microplate and the method for the immobilization of fused target proteins on magnetic beads for screening ligand mixtures.The high concentration of denatured protein potentially interfere with the malachite green assay of phosphate while the removal of denatured proteins is hampered for high-throughput screening approach based on improved malachite green assay of phosphate and microplate readers. Fortunately, at final levels of proteins<30mg/l, the improved malachite green assay of phosphate is resistant to denatured proteins, and thus the high-throughput screening approach is feasible when PDE isozymes have the specific acitivities> 0.008kU/g. The encoding sequence of human cyclic nucleotide phosphodiesterase 4B2 (hPDE4) and its truncated mutant (152aa-528aa) were inserted into the His-SUMO prokaryotic expression vector pReceiverl 3 for induced expression in Escherichia coli, which after purification through Ni2+-NTA column exhibited specific activities of>0.017kU/g (hPDE4) and>0.05 kU/g (the truncated mutant). By using Biotek ELX 800 microplate reader, affinities of two forms of PEDE4 for cAMP, rolipram and papaverine and were consistent with those by routine assay. Hence, the proposed method was effective for high-throughput-screening of inhibitors of phosphate-releasing enzymes.Rolipram is a classical lead of inhibitors of human PDE4; benzamides and its analogues had strong affinities to porcine aortic phosphodiesterase 4, were not tested with hPDE4. Benzamides of isovanillinic acid were synthesized from isovanillin via alkylation, oxidation, and amidation with aniline derivatives. Their purities were over 80% via HPLC. Their inhibition constants on hPDE4 as determined by the high-throughput screening approach supported their affinities, but gave a different structure-activity relationship in comparison to that with porcine enzyme PDE4.The screening of mixed inhibitors needs the selective immobilization of targets on magnetic particles.6His-tagged targets could be immobilized on Ni2+-NTA magnetic-submicron-particle (MSP) or bis-sulfone-functionalized MSP. Ni2+-NTA-MSP is more readily-available than bis-sulfone-functionalized MSP. The noncovalent conjugates of 6His-tagged targets and Ni2+-NTA-MSP are stable at pH 8.0, while pH 7.4 is suitable for determining ligand affinities and pH may affect ligand affinities on targets. The inhibition potency of tested compounds was indeed different at pH 7.4 and 8.0. The noncovalent conjugate of 6His-tagged Escherichia coli alkaline phosphatase and Ni2+-NTA-MSP released less than 6% of the immobilized enzyme in 3 h at pH 7.4, which enabled the recovery of> 90% target-ligand complexes from mixtures of competitive binding reaction. For a 6His-tagged truncated mutant of hPDE4 and Ni2+-NTA-MSP, the noncovalent conjugate showed similar stability at pH 7.4. After immobilization on Ni2+-NTA-MSP and bis-sulfone-functionalized MSP, these two 6His-tagged enzymes reserved>90% of their original activities. The use of bis-sulfone-MSP caused nonspecific adsorption of those two 6His-tagged enzymes and much longer time for immobilization. Therefore, it was feasible to use Ni2+-NTA-MSP for site-specific immobilization of 6His-tagged targets to screen ligands in mixtures. |
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