Development of a recombinant cell-based system for the characterisation of phosphodiesterase 4 isoforms and evaluation of inhibitors.
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.
Allen
R A
RA
Celltech Therapeutics Ltd., Slough, Berkshire, UK. rallen@celltech.co.uk
Merriman
M W
MW
Perry
M J
MJ
Owens
R J
RJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002384:Catalysis; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D007545:Isoproterenol / Q000494:pharmacology; D018343:Receptors, Adrenergic, beta-2 / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics; D013997:Time Factors; D014162:Transfection
D000318:Adrenergic beta-Agonists; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D007545:Isoproterenol