Implications of PDE4 Structure on Inhibitor Selectivity Across PDE Families

Home , Cilomilast, PDE3 inhibitor, Rolipram, Zardaverine

Implications of PDE4 structure on inhibitor selectivity across PDE families

HKe1*

1Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Phosphodiesterases (PDEs) control cellular concentrations of cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP). PDE4 and PDE5 selectively hydrolyze cAMP and cGMP, respectively. PDE family members share approximately 25% sequence identity within a conserved catalytic domain of about 300 amino acids. Crystal structure analysis of PDE4’s catalytic domain identifies two metal-binding sites: a high-affinity site and a low-affinity site, which probably bind zinc (Zn2 þ )andmagnesium(Mg2 þ ), respectively. Absolute conservation among the PDEs of two histidine and two aspartic acid residues for divalent metal binding suggests the importance of these amino acids in catalysis. Although active sites of PDEs are apparently structurally similar, PDE4 is specifically inhibited by selective inhibitors such as rolipram, while PDE5 is preferentially blocked by sildenafil. Modeling interactions of the PDE5 inhibitor sildenafil with the PDE4 active site may help explain inhibitor selectivity and provide useful information for the design of new inhibitors. International Journal of Impotence Research (2004) 16, S24–S27. doi:10.1038/sj.ijir.3901211

Keywords: phosphodiesterase inhibitors; PDE4; sildenafil; rolipram; 30,50-cyclic-nucleotide phosphodiesterase; protein conformation; binding sites

Introduction The three subdomains jointly form a deep pocket. This pocket contains 12 of the 17 residues absolutely conserved across all PDE families. It is the In all, 11 families of phosphodiesterase (PDE) have only pocket on the enzyme with sufficient volume been identified in the human body. Each family of (about 450 A˚ 3) to accommodate a molecule with the PDE exhibits a preference to either hydrolyze cyclic size of cAMP (about 250 A˚ 3), and thus must serve as adenosine monophosphate (cAMP) or cyclic guano- the catalytic pocket. sine monophosphate (cGMP) to adenosine mono- Helix 17 (residue 496–508) interacts with the phosphate (AMP) or to guanosine monophosphate residues of the catalytic pocket via a crystallo- (GMP), respectively. However, all 11 PDE families graphic symmetry in the crystal of PDE4B. However, contain the conserved catalytic domain of approxi- this helix exists in a random conformation within mately 300 amino acids, implying similar three- the structure of PDE4D2.2 The conformational dimensional structures of the catalytic domains. variation of the C-terminal residues in the PDE4 The crystal structure of the catalytic domain of family as well as the sequence diversity in the 11 PDE4B (residues 152–528) revealed 17 alpha he- PDE families may suggest a role for the C-terminus 1 lices, which form three subdomains. Subdomain 1, in the regulation of PDE hydrolytic activity. The or the N-terminal domain, is composed of a group of sequence alignment of PDEs identified two con- seven a helices (H1–H7). Subdomain 2 is composed served Hx3Hx24–26E sequences that are characteristic of four a helices (H8–H11). Subdomain 3 consists of motifs for zinc binding,3,4 thus suggesting that PDE five a helices (H12–H16) and an extended loop is a zinc enzyme. Indeed, biochemical studies have forming a b hairpin between H12 and H13. shown that a divalent metal is required for the catalytic activity of PDE.5 Among the divalent metals, Mg2 þ ,Co2 þ ,Mn2 þ , Ni2 þ , as well as Zn2 þ at low concentration, serve as *Correspondence: H Ke, PhD, Department of Biochemistry and Biophysics, University of North Carolina at Chapel the catalytic metal. However, the number of zinc atoms required for binding has sparked controversy Hill, 320 Mary E Jones Building, Chapel Hill, NC 27599- 2 þ 7260, USA. among researchers: One Zn site per PDE4A 6 2 þ 7 E-mail: [email protected] monomer two Zn for Vibrio fischeri PDE and PDE4 structure and inhibitor selectivity HKe S25 for PDE4A,8 and three Zn2 þ for PDE5.9 The crystal structure has revealed that the active site of PDE4 contains a cluster of two metal atoms separated by about 3.9 A˚ ,1 and coordinates with two histidines and two aspartic acids. Diverging from the prediction that each Hx3Hx24–26E motif binds a metal ion, two Hx3Hx24–26E motifs jointly interact with two metal sites simultaneously. Since other motifs unlikely exist for the binding of divalent metals, two metals should apply to all families of PDE, as evidenced by the molecule’s crystal structure. The first metal atom sits at the bottom of the catalytic pocket and coordinates with two histidines (H164, H200 in PDE4D2) and two aspartic acids (Asp201 and Asp318). As these metal binding residues belong, respectively, to the three PDE4 subdomains, the apparent role of the metal is to stabilize the protein structure. On the other hand, recent study on the PDE4–AMP structure showed that the metal also interacts with the phosphate oxygen and thus strongly suggests a catalytic role.10 The strong anomalous scattering at the wavelength of the zinc absorption edge suggests the first metal is a zinc, and is consistent with the fact that zinc, at low concentration, activates the enzyme.9 In PDE4D2, the second metal ion coordinates with D318, a bound water, Figure 1 Ribbon presentation of the PDE4 catalytic domain. and possibly with the phosphate group of the substrate. Owing to the weak binding of the site, the type of the second divalent metal is difficult to assign, although magnesium (Mg) or manganese The FDA has approved several PDE inhibitors as (Mn) is likely, according to biochemical studies therapeutic agents for human diseases. For example, (Figure 1). sildenafil (Viagras) is a drug for male erectile On the basis of the observations that all metal- dysfunction, and the PDE3 inhibitor cilostazol is a binding residues are absolutely conserved in drug utilized in the treatment of intermittent the PDE families, and that cAMP and cGMP contain claudication. However, it remains a mystery how a common phosphate group for the hydrolysis, selective PDE inhibitors that possess different a universal mechanism can be proposed for the chemical structures bind to apparently similar hydrolysis of cAMP or cGMP by all families of catalytic pockets. PDE. Thus, two metal ions hold the phosphate Rolipram, an antidepressant available in Europe group of cAMP in the active site and polarize and Japan, selectively inhibits members of the PDE4 the phosphor–oxygen bond to make the phosphor family. In fact, sensitivity to rolipram is considered a atom partially positively charged. The histidine defining feature of this PDE family. Rolipram, (His160 in PDE4D2) that is absolutely conserved cilomilast, and other inhibitors of PDE4 are thought in all families of PDE would protonate the O30 to act by direct competition with the substrate.13 group to further polarize the P–O bond for an They selectively inhibit PDE4 and increase the easy nucleophilic attack. Finally, a hydroxide accumulation of cAMP. The degree of inhibition ion on water molecule that is bound to the varies with the PDE4 subtypes. A direct correlation metals and activated by aspartic acid attacks has been observed between the net loss of protein nucleophilically the phosphor atom to facilitate residue interactions and the degree of rolipram the catalysis. resistance in mutations. The positions of several Each PDE family possesses its own inhibitors that drug sensitivity-determinant residues define a sur- either weakly inhibit or do not crossly inhibit other face leading to the substrate- and drug-binding sites. PDE families. Selective inhibitors of PDEs have been This suggests that an approach channel leads to the rapidly identified and widely studied as cardiotonic active site. agents, vasodilators, smooth muscle relaxants, anti- Recently, the crystal structure of PDE4D, in depressants, antithrombotic compounds, antiasthma complex with the inhibitor zardaverine, has been compounds, and agents for improving cognitive reported.14 Zardaverine binds to the hydrophobic functions, such as memory.11,12 pocket of the active site in a mode of competition

International Journal of Impotence Research PDE4 structure and inhibitor selectivity HKe S26 with the substrate cAMP, but does not directly facilitate discovery of highly selective inhibitors interact with the metals. It forms two hydrogen for each family of PDEs and thus dramatically bonds with Gln369 and interacts with residues improve the side-effect profile of PDE inhibitors. Met273, Leu319, Ile336, Met337, Phe340, and A molecular basis for the side effects of drugs Phe372 (PDE4D2 numbering). The fact that zarda- is their binding to nontargeted molecules. This is verine is a dual inhibitor of PDE3 and PDE415 especially important because the human body implies that the structural information of the active contains over 60 isoforms of PDE. The substrate site of PDE4 may be useful for design of not only specificity and the inhibitor selectivity of the PDE PDE4 selective inhibitors, but also of other PDE families are two essential areas for further research selective inhibitors. on PDE inhibitors. To investigate selectivity, the PDE5 selective An outstanding question is whether inhibitor inhibitor sildenafil (Viagras) was docked into the selectivity is determined by a few principal resi- catalytic pocket of PDE4D2 (Figure 2). Sildenafil has dues, or by subtle conformational differences at the the IC50 value of approximately 1 nM against PDE5, active site of PDE, or by a combination of both. and about 1000-fold selectivity of PDE5 over Although the PDE4 structures have clarified inhi- PDE4.16 The model demonstrated an overall well bitor selectivity to some degree, a thorough under- fit of sildenafil into the catalytic pocket of PDE4. standing of this phenomenon requires analysis of Thus, the purine-like ring of sildenafil sits in the the molecular configurations of other PDE families. hydrophobic pocket and forms a hydrogen bond, An additional unanswered issue hinges on the respectively, with Gln369 and Tyr159. However, development of selective inhibitors against subtypes Asn321, a residue showing dramatic variation of a PDE family. The crystal structures of PDE4, in across PDE families, collides with sildenafil. both their catalytic and regulatory domains, repre- On the basis of this model, alanine in PDE5, sent powerful tools for the design of subtype- which corresponds to Asn321, is expected to make selective inhibitors and a future direction for PDE room for hydrophobic interactions with sildenafil. inhibitor development. Thus, the same Asn to Ala mutation in PDE6 and PDE11 may explain their good sildenafil selectivity. The crystal structure of the catalytic domain of PDE4 provided valuable information on the active site of PDE4, and a potential guideline for design of References subtype selective inhibitors toward PDE4. This knowledge may also influence future development 1XuRX et al. Atomic structure of PDE4: insights into of drugs selective to other PDEs, as shown by the phosphodiesterase mechanism and specificity. Science 2000; docking of sildenafil into the PDE4 active site. 288: 1822–1825. Structure-based drug design may promote and 2 Huai Q et al. Three dimensional structures of PDE4D in complex with roliprams and implication on inhibitor selectivity. Structure 2003; 11: 865–873. 3 Vallee BL, Auld DS. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 1990; 29: 5647–5659. 4 Wilcox DE. Binuclear metallohydrolases. Chem Rev 1996; 96: 2435–2458. 5 Hardman JG et al. The formation and metabolism of cyclic GMP. Ann NY Acad Sci 1971; 185: 27–35. 6 Percival MD, Yeh B, Falgueyret JP. Zinc dependent activation of cAMP-specific phosphodiesterase (PDE4A). Biochem Bio- phys Res Commun 1997; 241: 175–180. 7 Callahan SM, Cornell NW, Dunlap PV. Purification and properties of periplasmic 30,50-cyclic nucleotide phosphodiesterase. A novel zinc-containing enzyme from the marine symbiotic bacterium Vibrio fischeri. J Biol Chem 1995; 270: 17627–17632. 8 Omburo GA, Jacobitz S, Torphy TJ, Colman RW. Critical role of conserved histidine pairs HNXXH and HDXXH in recombi- nant human phosphodiesterase 4A. Cell Signal 1998; 10: 491–497. 9 Francis SH, Colbran JL, McAllister-Lucas LM, Corbin JD. Zinc interactions and conserved motifs of the cGMP-binding cGMP- specific phosphodiesterase suggest that it is a zinc hydrolase. J Biol Chem 1994; 269: 22477–22480. 10 Huai Q, Colicelli J, Ke H. The crystal structure of AMP-bound PDE4 suggests a mechanism for phosphodiesterase catalysis. Biochemistry 2003; 42: 13220–13226. Figure 2 Modeling of sildenafil binding to the active site of PDE4: 11 Huang Z, Ducharme Y, Macdonald D, Robichaud A. The next Y ¼ Tyrosine; H ¼ Histidine; M ¼ Methionine; N ¼ Asparagine; generation of PDE4 inhibitors. Curr Opin Chem Biol 2001; 5: T ¼ Threonine; F ¼ Phenylalanie. 432–438.

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