title: Phosphodiesterase 4 conformers: preparation of recombinant enzymes and assay for inhibitors.
abstract: Cyclic nucleotides are key regulators of many cellular processes. Their immediate action is terminated through the activity of phosphodiesterases, a diverse family of enzymes. This diversity has given rise to drug discovery opportunities, and assay technology is therefore of key importance. Inhibitors of the cyclic-AMP-specific phosphodiesterases (the PDE4 family) are drug candidates for a variety of inflammatory disorders. However, PDE4 inhibitors, besides their immunomodulatory effects, also cause side effects including nausea and emesis. Recently, it has been suggested that PDE4 exists in two different conformations with respect to inhibition by the prototypical compound rolipram. Inhibition of the low-affinity conformer is thought to give rise to anti-inflammatory effects, and inhibition of the high-affinity conformer to side effects. Therefore, a selective inhibitor of the low-affinity conformer may have clinical utility. Methods are described to prepare recombinant forms of PDE4B that allow screening for compounds that could preferentially inhibit the low-affinity conformer. Furthermore, conditions for an efficient, scintillation proximity, microtiter plate-based assay are described, providing a considerable advance over previous assays in terms of throughput and automatability.
authors.affiliation: Eisai London Research Laboratories Ltd, Bernard Katz Building, University College London, Gower Street, London, WC1E 6BT, United Kingdom.
authors.forename: C
authors.initials: C
authors.lastname: Bardelle
authors.forename: C
authors.initials: C
authors.lastname: Smales
authors.forename: M
authors.initials: M
authors.lastname: Ito
authors.forename: K
authors.initials: K
authors.lastname: Nomoto
authors.forename: E Y
authors.initials: EY
authors.lastname: Wong
authors.forename: H
authors.initials: H
authors.lastname: Kato
authors.forename: T
authors.initials: T
authors.lastname: Saeki
authors.forename: J M
authors.initials: JM
authors.lastname: Staddon
mesh_terms: D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000235:genetics; D001331:Automation; D001483:Base Sequence; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000032:analysis* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D020889:Rolipram / Q000032:analysis* / Q000494:pharmacology
chemical_list: D017931:DNA Primers; D010726:Phosphodiesterase Inhibitors; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517360:PDE4B protein, human; D020889:Rolipram