Differential inhibitor sensitivity between human recombinant and native photoreceptor cGMP-phosphodiesterases (PDE6s).
Human photoreceptor cGMP-phosphodiesterases (PDE6s) are important reagents in PDE inhibitor discovery. However, recombinant human PDE6s have not been expressed, and isolation of native human PDE6s is highly difficult. In this study, the catalytic subunit(s) of human rod and cone PDE6s (PDE6alphabeta and PDE6alpha', respectively) were co-expressed or expressed separately as catalytically active enzymes. Sildenafil inhibited both the recombinant PDE6s in a dose-dependent manner with Ki values of 94 and 98 nM, respectively. These Ki values were four-fold higher than that (25 nM) of a human native PDE6 preparation. Similarly, 3-isobutyl-1-methylxanthine (IBMX)'s Ki values for the recombinant PDE6s were five- to eight-fold higher than that of the native enzyme. However, E4021 and zaprinast exhibited much (30-80-fold) lower potencies for the recombinant PDE6s than for the native enzyme. Additional PDE5 inhibitors representing other structural classes and possessing different selectivity against native PDE6 also showed different potencies against the recombinant and native PDE6s. In particular, one class of xanthine analogues exhibited significantly (5-15-fold) higher potencies for the recombinant PDE6s than for the native enzyme. Our data demonstrates that the recombinant and native PDE6s exhibit differential sensitivity to inhibitors, and cautions the use of recombinant catalytic subunits of PDE6 in drug discovery or in structural/functional studies.
Zhang
Ji
J
Allergy Department, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA.
Kuvelkar
Reshma
R
Wu
Ping
P
Egan
Robert W
RW
Billah
M Motasim
MM
Wang
Peng
P
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D006801:Humans; D007313:Insecta / Q000166:cytology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D010786:Photoreceptor Cells / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology*; D011687:Purines; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors*; D000068677:Sildenafil Citrate; D013450:Sulfones; D014162:Transfection
D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D011994:Recombinant Proteins; D013450:Sulfones; D000068677:Sildenafil Citrate; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; C517431:PDE6B protein, human