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We described the development of a recombinant cell-based system for the characterisation of phosphodiesterase (PDE) 4 isoforms and the evaluation of inhibitors. The Chinese hamster ovary (CHO) cell, which was found to have a low endogenous PDE4 background and no beta-adrenergic receptors (beta-AR), was transiently transfected with beta-AR and various PDE4 isoforms which were expressed as functionally coupled molecules. From correlations of elevation of adenosine 3',5'-cyclic monophosphate in situ and the inhibition of catalytic activity in vitro with the various PDE4 isoforms, it was apparent that PDE4A4, 4B2, 4C2, 4D2, and 4D3 all adopted a high-affinity binding conformation (i.e. expressed the high-affinity rolipram binding site) in the CHO cell, whereas PDE4A330 was expressed in a low-affinity conformation in situ. This gives the opportunity of using this system to screen and optimise inhibitors against a low-affinity conformation of PDE4 in situ and use a high-affinity conformation of PDE4 as a counterscreen, as inhibitor activity against this conformer has been linked with undesirable side effects. This system could also be utilised to screen inhibitors against various PDE4 isoforms in isolation against a low endogenous PDE background in situ for isoform-selective inhibitors.

Type

Journal article

Journal

Biochem Pharmacol

Publication Date

15/06/1999

Volume

57

Pages

1375 - 1382

Keywords

3',5'-Cyclic-AMP Phosphodiesterases, Adrenergic beta-Agonists, Animals, CHO Cells, COS Cells, Catalysis, Cricetinae, Cyclic AMP, Cyclic Nucleotide Phosphodiesterases, Type 4, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical, Enzyme Activation, Enzyme Inhibitors, Humans, Isoenzymes, Isoproterenol, Receptors, Adrenergic, beta-2, Recombinant Proteins, Time Factors, Transfection