DUAL INHIBITION OF HUMAN TYPE-4 PHOSPHODIESTERASE ISOSTATES BY (R-ASTERISK,R-ASTERISK)-(+ -)-METHYL -METHOXYPHENYL]-3-METHYL-1-PYRROLIDINECARBOXYLATE/

Purified recombinant human type 4 phosphodiesterase B2B (HSPDE4B2B) ex ists in both a low-and a high-affinity state that bind (R)-rolipram wi th K-d'S Of ca. 500 and 1 nM, respectively [Rocque, W. J., Tian, G., W iseman, J. S., Holmes, W. D., Thompson, I. Z., Willard. D. Il., Patel, I. R., Wisely, G. B., Clay, W. C., Kadwell, S. H., Hoffman, C. R., an d Luther, M. A. (1997) Biochemistry 36, 14250-14361]. Since the tissue distribution of the two isostates may be significantly different, dev elopment of inhibitors that effectively inhibit both forms may be adva ntageous pharmacologically. In this study, enzyme inhibition and bindi ng of HSPDE4B2B by (R,R*)-(+/-)-methyl 4-methoxyphenyl]-3-methyl-1-py rrolidinecarboxylate (1), a novel inhibitor of phosphodiesterase 4 (PD E 4), were investigated Binding experiments demonstrated high-affinity binding of. to HSPDE4B2B with a stoichiometry of 1:1. Inhibition of P DE activity showed only a single transition with an observed K-i simil ar to the apparent K-d determined by the binding experiments. Deletion al mutants of HSPDE4B2B, which have been shown to bind (R)-rolipram wi th low affinity, were shown to interact with 1 with high affinity, ind istinguishable from the results obtained with the full-length enzyme. Bound 1 was completely displaced by (R)-rolipram, and the displacement showed a biphasic transition that resembles the biphasic inhibition o f HSPDE4B2B by (R)-rolipram. Theoretical analysis of the two transitio ns exemplified in the interaction of (R)-rolipram with HSPDE4B2B indic ated that the two isostates were nonexchangeable. Phosphorylation at s erines 487 and 489 on HSPDE4B2B had no effect on the stoichiometry of binding, the affinity for binding, or the inhibition of the enzyme by 1. These data further illustrate the presence of two isostates in PDE 4 as shown previously for (R)-rolipram binding and inhibition. In cont rast to (R)-rolipram, where only one of the two isostates of PDE 4 bin ds with high affinity, 1 is a potent, dual inhibitor of both of the is ostates of PDE 4. Kinetic and thermodynamic models describing the inte ractions between the nonexchangeable isostates of PDE 4 and its ligand s are discussed.