Intracellular localization of 7-benzylamino-6-chloro-2-piperazino-4-pyrrolidino-pteridine in membrane structures impeding the inhibition of cytosolic cyclic AMP-specific phosphodiesterase.
7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidino-pteridine (DC-TA-46) is a potent inhibitor of the rolipram-sensitive cAMP-specific phosphodiesterase isoenzyme family PDE4. DC-TA-46 inhibits cAMP-hydrolysis of PDE4 isolated from solid tumors of the human large cell lung tumor xenograft LXFL529 in the nanomolar range (IC(50)=16+/-5nM). Tumor cells, however, are growth inhibited only in the lower micromolar range as shown for the human large cell lung carcinoma cell line LXFL529L. To investigate reasons for the discrepancy between IC(50) values for target inhibition and inhibition of cell growth, uptake, subcellular distribution and elimination of the compound were measured. DC-TA-46 was rapidly taken up by the cells, predominantly localized in intracellular membranes. Elimination was slow, with 70% of the compound still persisting in the membranes 50hr after withdrawal. Confocal laser scanning microscopy showed a clear colocalization with a fluorescent marker for the endoplasmatic reticulum (ER). As a result of the subcellular localization, the membrane-bound PDE activity of LXFL529L cells was effectively inhibited by DC-TA-46 (IC(50)=0.06+/-0.02 microM). In contrast, inhibition of the cytosolic PDE activity was only achieved at concentrations >1 microM (IC(50)=2.0+/-0.5 microM), in the concentration range where also growth inhibition was observed. Thus, the inhibition of the intracellular PDE activity in the different cellular compartments appears to represent an important parameter for the evaluation of the inhibitory properties at least of this class of compounds.
Marko
Doris
D
Department of Chemistry, Division of Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Erwin-Schroedinger-Str. 52, D-67663, Kaiserslautern, Germany. marko@rhrk.uni-kl.de
Merz
Karl Heinz
KH
Kunz
Claudia
C
Müller
Anja
A
Tarasova
Nadya
N
Eisenbrand
Gerhard
G
D001692:Biological Transport; D000242:Cyclic AMP / Q000378:metabolism*; D006801:Humans; D010726:Phosphodiesterase Inhibitors / Q000493:pharmacokinetics*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010879:Piperazines / Q000493:pharmacokinetics*; D011621:Pteridines / Q000493:pharmacokinetics*; D013347:Subcellular Fractions; D014407:Tumor Cells, Cultured
D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011621:Pteridines; C081896:7-benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases