Molecular Docking of Competitive Phosphodiesterase Inhibitors ORLY DYM, IOANNIS XENARIOS, HENGMING KE, and JOHN COLICELLI University of California Los Angeles-Department of Energy Laboratory of Structural Biology and Molecular Medicine, University of California Los Angeles, Los Angeles, California (O.D., I.X.); Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (H.K.); and Department of Biological Chemistry and the Molecular Biology Institute, University of California Los Angeles School of Medicine, Los Angeles, California (J.C.) Received May 10, 2001; accepted September 25, 2001 This paper is available online at http://molpharm.aspetjournals.org ABSTRACT Mammalian phosphodiesterases types 3 and 4 (PDE3 and PDE4) hydrolyze cAMP and are essential for the regulation of this intracellular second messenger. These enzymes share structural and biochemical similarities, but each can be distin- guished by its sensitivity to isoenzyme-specific, substrate- competitive inhibitors. We present a model configuration for the PDE4 substrate (cAMP) and a PDE4-specific inhibitor (rolipram) within the active site of the enzyme. The docked models were also used to examine the structural consequences of mutations that confer resistance to rolipram and other PDE4-specific in- hibitors. The proposed rolipram-binding configuration is con- sistent with the substrate-competitive nature of inhibition and also provides a structural basis for the observed specificity of binding to the R- versus S-enantiomer. For mutations that render the enzyme rolipram-insensitive, there was generally an inverse relationship between the magnitude of the drug resis- tance and the distance of the altered residue from the predicted binding site. We observed a direct correlation between the net loss of protein residue interactions (van der Waals contacts and hydrogen bond interactions) and the degree of rolipram resis- tance. The positions of several drug sensitivity-determinant residues define a surface leading to the substrate- and drug- binding sites, suggesting a possible approach channel leading to the enzyme active site. The binding of other PDE4 inhibitors (high- and low-affinity) was also modeled and used to predict the involvement of residues that were not previously implicated in pharmacological interactions. cAMP is a ubiquitous intracellular second messenger. Its effects are principally mediated through the indirect activa- tion of cAMP-dependent protein kinases. There are families of enzymes that regulate cAMP synthesis (adenylyl cyclases) and cAMP degradation [phosphodiesterases (PDEs)] (Hous- lay and Milligan, 1997). The mammalian PDEs have been categorized into 11 types relating to their substrate selectiv- ity, kinetics, and pharmacological sensitivity (Francis et al., 2000; Soderling and Beavo, 2000). PDEs across families share considerable sequence similarity. Confined primarily to the catalytic domain, these conserved residues are likely to have direct or indirect involvement in cyclic nucleotide hy- drolysis (Jin et al., 1992; Jacobitz et al., 1997; Owens et al., 1997). Within PDE families, sequence identity is more exten- sive, reflecting greater relatedness in their biochemical prop- erties. PDEs are clinical targets for a range of biological disorders, such as congestive heart failure, asthma, chronic obstructive pulmonary disease, depression, retinal degrada- tion, and inflammation (Conti et al., 1991; Teixeira et al., 1997; Barnette and Underwood, 2000; Spina, 2000). There is a particular interest in the enzymes encoded by PDE4 genes (PDE4A, PDE4B, PDE4C, and PDE4D) in the search for therapeutically useful inhibitors (Raeburn et al., 1994; Co- han et al., 1996; Holbrook et al., 1996). Rolipram (4-[3-(cy- clopentyloxy)-4-methoxyphenyl]-2-pyrrolidinone) is one of the earliest and most extensively studied PDE4 inhibitors. It has an IC 50 of approximately 1 M (lower under some con- ditions) and shows at least a 100-fold selectivity for this PDE family. Indeed, sensitivity to rolipram has become a defining feature of these isozymes. As with other PDE inhibitors, enzymatic inhibition is competitive with substrate. Attempts to identify determinants of substrate- and inhibitor-binding within PDE4 have included a variety of deletion, chimera, and directed point-mutation studies (Jin et al., 1992; Pillai et al., 1993; Atienza et al., 1999). In addition, an approach of random mutagenesis coupled with rolipram-resistance selec- tion has been used to identify drug-specificity determinants within PDE4 (Pillai et al., 1993; Atienza et al., 1999). These mutant enzymes display different degrees of sensitivity to These studies were supported by National Institutes of Health Grants NS31911 (to J.C.), GM59791 (to H.K.), GM31299 (to O.D.), and United States Department of Energy Grant ER60615 (to I.X.). ABBREVIATIONS: PDE, phosphodiesterases; RP 73401, 3-cyclopentyloxy-N-(3,5-dichloropyridin-4-yl)-4-methoxybenzamide; IBMX, 3-isobutyl- 1-methylxanthine. 0026-895X/02/6120-20 –25$3.00 MOLECULAR PHARMACOLOGY Vol. 61, No. 20 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1096/951367 Mol Pharmacol 61:20–25, 2002 Printed in U.S.A. 20 at ASPET Journals on April 16, 2016 molpharm.aspetjournals.org Downloaded from