Novel inhibitors of the methicillin-resistant Staphylococcus aureus (MRSA)-pyruvate kinase.

Novel bisindolyl-cycloalkane indoles resulted from the reaction of aliphatic dialdehydes and indole. As bisindolyl-natural alkaloid compounds have recently been reported as inhibitors of the methicillin-resistant Staphylococcus aureus (MRSA)-pyruvate kinase (PK), we tested our novel compounds as MRSA PK inhibitors and now report first inhibiting activities. We discuss structure-activity relationships of structurally varied compounds. Activity influencing substituents have been characterized and relations to antibacterial activities of the most active compounds have been proved.

Synthetic analogues of the marine bisindole deoxytopsentin: potent selective inhibitors of MRSA pyruvate kinase.

As part of an ongoing study to elucidate the SAR of bisindole alkaloid inhibitors against the evolutionary conserved MRSA pyruvate kinase (PK), we present here the synthesis and biological activity of six dihalogenated analogues of the naturally occurring sponge metabolite deoxytopsentin, including the naturally occurring dibromodeoxytopsentin. The most active compounds displayed potent low nanomolar inhibitory activity against MRSA PK with concomitant significant selectivity for MRSA PK over human PK orthologues. Computational studies suggest that these potent MRSA PK inhibitors occupy a region of the small interface of the enzyme tetramer where amino acid sequence divergence from common human PK orthologues may contribute to the observed selectivity.

Discovery of a 1,2-bis(3-indolyl)ethane that selectively inhibits the pyruvate kinase of methicillin-resistant Staphylococcus aureus over human isoforms.

Methicillin-resistant Staphylococcus aureus pyruvate kinase (MRSA PK) has recently been identified as a target for development of novel antibacterial agents. Testing a series of 1,2-bis(3-indolyl)ethanes against MRSA PK has led to the discovery of a potent inhibitor that is selective over human isoforms.

Discovery and optimization of a new class of pyruvate kinase inhibitors as potential therapeutics for the treatment of methicillin-resistant Staphylococcus aureus infections.

A novel series of bis-indoles derived from naturally occurring marine alkaloid 4 were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK is not only critical for bacterial survival which would make it a target for development of novel antibiotics, but it is reported to be one of the most highly connected 'hub proteins' in MRSA, and thus should be very sensitive to mutations and making it difficult for the bacteria to develop resistance. From the co-crystal structure of cis-3-4-dihydrohamacanthin B (4) bound to S. aureus PK we were able to identify the pharmacophore needed for activity. Consequently, we prepared simple direct linked bis-indoles such as 10b that have similar anti-MRSA activity as compound 4. Structure-activity relationship (SAR) studies were carried out on 10b and led us to discover more potent compounds such as 10c, 10d, 10k and 10 m with enzyme inhibiting activities in the low nanomolar range that effectively inhibited the bacteria growth in culture with minimum inhibitory concentrations (MIC) for MRSA as low as 0.5 µg/ml. Some potent PK inhibitors, such as 10b, exhibited attenuated antibacterial activity and were found to be substrates for an efflux mechanism in S. aureus. Studies comparing a wild type S. aureus with a construct (S. aureus LAC Δpyk::Erm(R)) that lacks PK activity confirmed that bactericidal activity of 10d was PK-dependant.

Protein interaction networks as starting points to identify novel antimicrobial drug targets.

Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria. Current bacterial drug targets mainly consist of specific proteins or subsets of proteins without regard for either how these targets are integrated in cellular networks or how they may interact with host proteins. However, proteins rarely act in isolation, and the majority of biological processes are dependent on interactions with other proteins. Consequently, protein-protein interaction (PPI) networks offer a realm of unexplored potential for next-generation drug targets. In this review, we argue that the architecture of bacterial or host-pathogen protein interactomes can provide invaluable insights for the identification of novel antibacterial drug targets.

Optimization and structure-activity relationships of a series of potent inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as novel antimicrobial agents.

A novel series of hydrazones were synthesized and evaluated as inhibitors of methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase (PK). PK has been identified as one of the most highly connected 'hub proteins' in MRSA. PK has been shown to be critical for bacterial survival which makes it a potential target for development of novel antibiotics and the high degree of connectivity implies it should be very sensitive to mutations and thus less able to develop resistance. PK is not unique to bacteria and thus a critical requirement for such a PK inhibitor would be that it does not inhibit the homologous human enzyme(s) at therapeutic concentrations. Several MRSA PK inhibitors (including 8d) were identified using in silico screening combined with enzyme assays and were found to be selective for bacterial enzyme compared to four human PK isoforms (M1, M2, R and L). However these lead compounds did not show significant inhibitory activity for MRSA growth presumably due to poor bacterial cell penetration. Structure-activity relationship (SAR) studies were carried out on 8d and led us to discover more potent compounds with enzyme inhibiting activities in the low nanomolar range and some were found to effectively inhibit bacteria growth in culture with minimum inhibitory concentrations (MIC) as low as 1 µg/mL. These inhibitors bind in two elongated flat clefts found at the minor interfaces in the homo-tetrameric enzyme complex and the observed SAR is in keeping with the size and electronic constraints of these binding sites. Access to the corresponding sites in the human enzyme is blocked.

Cheminformatics-driven discovery of selective, nanomolar inhibitors for staphylococcal pyruvate kinase.

We have recently mapped the protein interaction network of methicillin-resistant Staphylococcus aureus (MRSA), which revealed its scale-free organization with characteristic presence of highly connected hub proteins that are critical for bacterial survival. Here we report the discovery of inhibitors that are highly potent against one such hub target, staphylococcal pyruvate kinase (PK). Importantly, the developed compounds demonstrate complete selectivity for the bacterial enzyme compared to all human orthologues. The lead 91nM inhibitor IS-130 has been identified through ligand-based cheminformatic exploration of a chemical space around micromolar hits initially generated by experimental screening. The following crystallographic study resulted in identification of a tetrameric MRSA PK structure where IS-130 is bound to the interface between the protein's subunits. This newly described binding pocket is not present in otherwise highly similar human orthologues and can be effectively utilized for selective inhibition of bacterial PK. The following synthetic modifications of IS-130, guided by structure-based molecular modeling, resulted in the development of MRSA PK inhibitors with much improved antimicrobial properties. Considering a notable lack of recent reports on novel antibacterial targets and cognate antibacterial compounds, this study provides a valuable perspective on the development of a new generation of antimicrobials. Equally noteworthy, the results of the current work highlight the importance of rigorous cheminformatics-based exploration of the results of high-throughput experiments.

Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as a target for bis-indole alkaloids with antibacterial activities.

Novel classes of antimicrobials are needed to address the emergence of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). We have recently identified pyruvate kinase (PK) as a potential novel drug target based upon it being an essential hub in the MRSA interactome (Cherkasov, A., Hsing, M., Zoraghi, R., Foster, L. J., See, R. H., Stoynov, N., Jiang, J., Kaur, S., Lian, T., Jackson, L., Gong, H., Swayze, R., Amandoron, E., Hormozdiari, F., Dao, P., Sahinalp, C., Santos-Filho, O., Axerio-Cilies, P., Byler, K., McMaster, W. R., Brunham, R. C., Finlay, B. B., and Reiner, N. E. (2011) J. Proteome Res. 10, 1139-1150; Zoraghi, R., See, R. H., Axerio-Cilies, P., Kumar, N. S., Gong, H., Moreau, A., Hsing, M., Kaur, S., Swayze, R. D., Worrall, L., Amandoron, E., Lian, T., Jackson, L., Jiang, J., Thorson, L., Labriere, C., Foster, L., Brunham, R. C., McMaster, W. R., Finlay, B. B., Strynadka, N. C., Cherkasov, A., Young, R. N., and Reiner, N. E. (2011) Antimicrob. Agents Chemother. 55, 2042-2053). Screening of an extract library of marine invertebrates against MRSA PK resulted in the identification of bis-indole alkaloids of the spongotine (A), topsentin (B, D), and hamacanthin (C) classes isolated from the Topsentia pachastrelloides as novel bacterial PK inhibitors. These compounds potently and selectively inhibited both MRSA PK enzymatic activity and S. aureus growth in vitro. The most active compounds, cis-3,4-dihyrohyrohamacanthin B (C) and bromodeoxytopsentin (D), were identified as highly potent MRSA PK inhibitors (IC(50) values of 16-60 nM) with at least 166-fold selectivity over human PK isoforms. These novel anti-PK natural compounds exhibited significant antibacterial activities against S. aureus, including MRSA (minimal inhibitory concentrations (MIC) of 12.5 and 6.25 µg/ml, respectively) with selectivity indices (CC(50)/MIC) >4. We also report the discrete structural features of the MRSA PK tetramer as determined by x-ray crystallography, which is suitable for selective targeting of the bacterial enzyme. The co-crystal structure of compound C with MRSA PK confirms that the latter is a target for bis-indole alkaloids. It elucidates the essential structural requirements for PK inhibitors in "small" interfaces that provide for tetramer rigidity and efficient catalytic activity. Our results identified a series of natural products as novel MRSA PK inhibitors, providing the basis for further development of potential novel antimicrobials.

Identification of pyruvate kinase in methicillin-resistant Staphylococcus aureus as a novel antimicrobial drug target.

Novel classes of antimicrobials are needed to address the challenge of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Using the architecture of the MRSA interactome, we identified pyruvate kinase (PK) as a potential novel drug target based upon it being a highly connected, essential hub in the MRSA interactome. Structural modeling, including X-ray crystallography, revealed discrete features of PK in MRSA, which appeared suitable for the selective targeting of the bacterial enzyme. In silico library screening combined with functional enzymatic assays identified an acyl hydrazone-based compound (IS-130) as a potent MRSA PK inhibitor (50% inhibitory concentration [IC50] of 0.1 µM) with >1,000-fold selectivity over human PK isoforms. Medicinal chemistry around the IS-130 scaffold identified analogs that more potently and selectively inhibited MRSA PK enzymatic activity and S. aureus growth in vitro (MIC of 1 to 5 µg/ml). These novel anti-PK compounds were found to possess antistaphylococcal activity, including both MRSA and multidrug-resistant S. aureus (MDRSA) strains. These compounds also exhibited exceptional antibacterial activities against other Gram-positive genera, including enterococci and streptococci. PK lead compounds were found to be noncompetitive inhibitors and were bactericidal. In addition, mutants with significant increases in MICs were not isolated after 25 bacterial passages in culture, indicating that resistance may be slow to emerge. These findings validate the principles of network science as a powerful approach to identify novel antibacterial drug targets. They also provide a proof of principle, based upon PK in MRSA, for a research platform aimed at discovering and optimizing selective inhibitors of novel bacterial targets where human orthologs exist, as leads for anti-infective drug development.

Mapping the protein interaction network in methicillin-resistant Staphylococcus aureus.

Mortality attributable to infection with methicillin-resistant Staphylococcus aureus (MRSA) has now overtaken the death rate for AIDS in the United States, and advances in research are urgently needed to address this challenge. We report the results of the systematic identification of protein-protein interactions for the hospital-acquired strain MRSA-252. Using a high-throughput pull-down strategy combined with quantitative proteomics to distinguish specific from nonspecific interactors, we identified 13,219 interactions involving 608 MRSA proteins. Consecutive analyses revealed that this protein interaction network (PIN) exhibits scale-free organization with the characteristic presence of highly connected hub proteins. When clinical and experimental antimicrobial targets were queried in the network, they were generally found to occupy peripheral positions in the PIN with relatively few interacting partners. In contrast, the hub proteins identified in this MRSA PIN that are essential for network integrity and stability have largely been overlooked as drug targets. Thus, this empirical MRSA-252 PIN provides a rich source for identifying critical proteins essential for network stability, many of which can be considered as prospective antimicrobial drug targets.

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from methicillin-resistant Staphylococcus aureus.

Novel antimicrobial targets are urgently needed to overcome rising antibiotic resistance of important human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Here we report the essentiality and kinetic properties of MRSA pyruvate kinase (PK). Targetron-mediated gene disruption demonstrated PK is essential for S. aureus growth and survival, suggesting that this protein may be a potential drug target. The presence of the pfk (6-phosphofructokinase)-pyk operon in MRSA252, and the nonessential nature of PFK shown by targetron, further emphasized the essential role of PK in cell viability. The importance of PK in bacterial growth was confirmed by showing that its enzymatic activity peaked during the logarithmic phase of S. aureus growth. PK from Staphylococcus and several other species of bacteria have an extra C-terminal domain (CT) containing a phosphoenolpyruvate (PEP) binding motif. To elucidate the possible structure and function of this sequence, the quaternary structures and kinetic properties of the full-length MRSA PK and truncated MRSA PK lacking the CT domain were characterized. Our results showed that (1) MRSA PK is an allosteric enzyme with homotetramer architecture activated by AMP or ribose 5-phosphate (R5P), but not by fructose 1,6-bisphosphate (FBP), which suggests a different mode of allosteric regulation when compared with human isozymes, (2) the CT domain is not required for the tetramerization of the enzyme; homotetramerization occurred in a truncated PK lacking the domain, (3) truncated enzyme exhibited high affinity toward both PEP and ADP and exhibited hyperbolic kinetics toward PEP in the presence of activators (AMP and R5P) consistent with kinetic properties of full-length enzyme, indicating that the CT domain is not required for substrate binding or allosteric regulation observed in the holoenzyme, (4) the kinetic efficiency (k(cat)/S(0.5)) of truncated enzyme was decreased by 24- and 16-fold, in ligand-free state, toward PEP and ADP, respectively, but was restored by 3-fold in AMP-bound state, suggesting that the sequence containing the CT domain (Gly(473)-Leu(585)) plays a substantial role in enzyme activity and comformational stability, and (5) full-length MRSA PK activity was stimulated at low concentrations of ATP (e.g., 1 mM) and inhibited by inorganic phosphate and high concentrations of FBP (10 mM) and ATP (e.g., >2.5 mM), whereas for truncated enzyme, stimulation at low concentrations of ATP was lost. These findings suggest that the CT domain is involved in maintaining the specificity of allosteric regulation of MRSA PK by AMP, R5P, and ATP. The CT extension also encodes a protein domain with homology to enzyme I of the Escherichia coli sugar-PTS system, suggesting that MRSA PK may also exert an important regulatory role in sugar transport metabolism. These findings yield new insights into MRSA PK function and mode of allosteric regulation which may aid in the development of clinically important drugs targeting this enzyme and further define the role of the extra C-terminal domain in modulating the enzyme's activity.

Allosteric-site and catalytic-site ligand effects on PDE5 functions are associated with distinct changes in physical form of the enzyme.

Native phosphodiesterase-5 (PDE5) homodimer contains distinct non-catalytic cGMP allosteric sites and catalytic sites for cGMP hydrolysis. Purified recombinant PDE5 was activated by pre-incubation with cGMP. Relatively low concentrations of cGMP produced a Native PAGE gel shift of PDE5 from a single band position (lower band) to a band with decreased mobility (upper band); higher concentrations of cGMP produced a band of intermediate mobility (middle band) in addition to the upper band. Two point mutations (G659A and G659P) near the catalytic site that reduced affinity for cGMP substrate retained allosteric cGMP-binding affinity like that of WT PDE5 but displayed cGMP-induced gel shift only to the middle-band position. The upper band could represent a form produced by cGMP binding to the catalytic site, while the middle band could represent a form produced by cGMP binding to the allosteric site. Millimolar cGMP was required for gel shift of PDE5 when added to the pre-incubation before Native PAGE, presumably due to removal of most of the cGMP during electrophoresis, but micromolar cGMP was sufficient for this effect if cGMP was included in the native gel buffer. cGMP-induced gel shift was associated with stimulation of PDE5 catalytic activity, and the rates of onset and reversibility of this effect suggested that it was due to cGMP binding to the allosteric site. Incubation of PDE5 with non-hydrolyzable, catalytic site-specific, substrate analogs such as the inhibitors sildenafil and tadalafil, followed by dilution, did not produce activation of catalytic activity like that obtained with cGMP, although both inhibitors produced a similar gel shift to the upper band as that obtained with cGMP. This implied that occupation of the catalytic site alone can produce a gel shift to the upper band. PDE5 activation or gel shift was reversed by lowering cGMP with dilution followed by at least 1h of incubation. Such slow reversibility could prolong effects of cGMP on PDE5 in cells after decline of this nucleotide. Reversal was also achieved by Mg(++) addition to the pre-incubation mixture to promote cGMP degradation, but Mg(++) addition did not reverse the gel shift caused by sildenafil, which is not hydrolyzed by PDE5. Upon extensive dilution, the effect of tadalafil, a potent PDE5 inhibitor, to enhance catalytic-site affinity for this inhibitor was rapidly reversed. Thus, kinetic effect of binding of a high-affinity PDE5 inhibitor to the catalytic site is more readily reversible than that obtained by cGMP binding to the allosteric site. It is concluded that cGMP or PDE5 inhibitor binding to the catalytic site, or ligand binding to both the catalytic site and allosteric site simultaneously, changes PDE5 to a similar physical form; this form is distinct from that produced by cGMP binding to the allosteric site, which activates the enzyme and reverses more slowly.

Phosphorylation increases affinity of the phosphodiesterase-5 catalytic site for tadalafil.

Phosphodiesterase-5 (PDE5) is phosphorylated at a single serine residue by cyclic nucleotide-dependent protein kinases. To test for a direct effect of phosphorylation on the PDE5 catalytic site, independent of cGMP binding to the allosteric sites of the enzyme, binding of the catalytic site-specific substrate analog [(3)H]tadalafil to PDE5 was measured. Phosphorylation increased [(3)H]tadalafil binding 3-fold, whereas cGMP caused a 1.6-fold increase. Combination of both treatments caused more than 4-fold increase in [(3)H]tadalafil binding, and effects were additive only at submaximal stimulation. Consistent with the increase in affinity, phosphorylation slowed the [(3)H]tadalafil exchange-dissociation rate from PDE5 more than 6-fold. Finally, phosphorylation increased affinity for hydrolysis of a catalytic site-specific cGMP analog, 2'-O-anthraniloyl-cGMP, by approximately 3-fold. The combined results showed that phosphorylation activates PDE5 catalytic site independently of cGMP binding to the allosteric sites. The results suggested that phosphorylation acts in concert with allosteric cGMP binding to stimulate the PDE5 catalytic site, which should promote negative feedback regulation of the cGMP pathway in intact cells. By increasing the affinity of the catalytic site, phosphorylation should also consequently increase the potency and duration of PDE5 inhibitor action.

Critical amino acids in phosphodiesterase-5 catalytic site that provide for high-affinity interaction with cyclic guanosine monophosphate and inhibitors.

The molecular bases for phosphodiesterase 5 (PDE5) catalytic-site affinity for cyclic guanosine monophosphate (cGMP) and potency of inhibitors are poorly understood. Cocrystal structures of PDE5 catalytic (C) domain with inhibitors reveal a hydrogen bond and hydrophobic interactions with Tyr-612, hydrogen bonds with Gln-817, a hydrophobic clamp formed by Phe-820 and Val-782, and contacts with His-613, Leu-765, and Phe-786 [Sung et al. (2003) Nature 425, 98-102; Huai et al. (2004) J. Biol. Chem. 279, 13095-13101]. Present results of point mutations of full-length PDE5 showed that maximum catalysis was decreased 2650-fold in H613A and 55-fold in F820A. Catalytic-site affinities for cGMP, vardenafil, sildenafil, tadalafil, or 3-isobutyl-1-methylxanthine (IBMX) were respectively weakened 14-, 123-, 30-, 51-, and 43-fold for Y612A; 63-, 511-, 43-, 95- and 61-fold for Q817A; and 59-, 448-, 71-, 137-, and 93-fold for F820A. The data indicate that these three amino acids are major determinants of affinity for cGMP and potency of selective and nonselective inhibitors, and that higher vardenafil potency over sildenafil and tadalafil results from stronger contacts with Tyr-612, Gln-817, and Phe-820. Affinity of V782A for cGMP, vardenafil, sildenafil, tadalafil, or IBMX was reduced 5.5-, 23-, 10-, 3-, and 12-fold, respectively. Change in affinity for cGMP, vardenafil, sildenafil, or IBMX in Y612F, H613A, L765A, or F786A was less, but affinity of H613A or F786A for tadalafil was weakened 37- and 17-fold, respectively. The results quantify the role of PDE5 catalytic-site residues for cGMP and inhibitors, indicate that Tyr-612, Gln-817, and Phe-820 are the most important cGMP or inhibitor contacts studied, and identify residues that contribute to selectivity among different classes of inhibitors.

Conversion of phosphodiesterase-5 (PDE5) catalytic site to higher affinity by PDE5 inhibitors.

Phosphodiesterase-5 (PDE5) specifically hydrolyzes cGMP, thereby contributing to modulation of intracellular levels of this nucleotide. In the present study, preincubation with cGMP increased PDE5 catalytic activity for cGMP degradation, and it converted the PDE5 catalytic site to a form that was more potently inhibited by each of the three PDE5 catalytic site-specific inhibitors: sildenafil, vardenafil, and tadalafil. These results implied that elevated cGMP initiates a physiological negative feedback on the cGMP pathway by increasing the affinity of the PDE5 catalytic site for cGMP. This increase in catalytic site activity or affinity for inhibitors could be caused by binding of cGMP to either the PDE5 allosteric sites, catalytic site, or both. Whether occupation of the catalytic site alone could mediate the effect was examined using radiolabeled PDE5 inhibitors in the absence of cGMP. Exchange-dissociation of [(3)H]sildenafil (Viagra), [(3)H]vardenafil (Levitra), or [(3)H]tadalafil (Cialis) from full-length PDE5 or isolated catalytic domain revealed two kinetic components (slow and fast). Extended preincubation of full-length PDE5, but not isolated catalytic domain, with (3)H inhibitors converted the biphasic pattern to a single slow (high-affinity) component. Studies of amino-terminally truncated PDE5 established that full-length mammalian GAF-B (cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlA) subdomain conjoined with the catalytic domain was sufficient for this conversion. In conclusion, binding of substrate or substrate analogs such as PDE5 inhibitors to the catalytic site converts a fast (low-affinity) inhibitor dissociation component of the PDE5 catalytic site to a slow (high-affinity) inhibitor dissociation component. This effect is predicted to improve the substrate affinity or inhibitory potencies of these compounds in intact cells.

N-Terminal domain of phosphodiesterase-11A4 (PDE11A4) decreases affinity of the catalytic site for substrates and tadalafil, and is involved in oligomerization.

The phosphodiesterase-11A (PDE11) family consists of four splice variants (PDE11A1-PDE11A4) that contain a conserved carboxyl-terminal (C-terminal) catalytic domain that hydrolyzes cAMP and cGMP; the amino-termini (N-termini) vary in length and amino acid sequence. PDE11A2, PDE11A3, and PDE11A4 contain one or more GAF (cGMP-binding phosphodiesterase, Anabaena adenylyl cyclase, and Escherichia coli FhlA) subdomains. In the present study, PDE11A1 and PDE11A2 demonstrated higher affinity for cAMP and cGMP when directly compared to that of the longest isoform, PDE11A4. Moreover, PDE11A3, PDE11A2, and PDE11A1, which contain progressively shorter N-termini, were more sensitive than PDE11A4 to inhibition by two structurally unrelated inhibitors, tadalafil (Cialis) and vardenafil (Levitra). The substrate and inhibitor affinity differences among the PDE11 isozymes could not be ascribed to differences in their quaternary structure since PDE11A4, PDE11A3, and PDE11A2 were determined to be dimers, and PDE11A1 was a tetramer. These data also demonstrate that PDE11 isozymes containing at least 123 C-terminal amino acids of the GAF-B domain are stable oligomers and that GAF-A is not required for oligomerization. The isolated PDE11 catalytic domain (Met-563-Asn-934) displayed both monomeric and dimeric forms, and upon dilution, this domain was primarily monomeric, indicating that the main oligomerization contacts are within the N-termini of PDE isozymes. This report is the first to describe an inhibitory effect of the N-terminal region of PDE11A4 on the affinity of the catalytic domain for both substrates and inhibitors and the first to define the quaternary structure and the regions that contribute to this structure within the human PDE11A family.

Phosphorylation of phosphodiesterase-5 is promoted by a conformational change induced by sildenafil, vardenafil, or tadalafil.

Phosphodiesterase-5 (PDE5) inhibitors (sildenafil, vardenafil, or tadalafil) or phosphorylation by cyclic nucleotide-dependent protein kinase causes an apparent conformational change in PDE5, as indicated by a shift in migration on non-denaturing PAGE gels and an altered pattern of tryptic digestion. Combination of cGMP and a PDE5 inhibitor or phosphorylation does not cause a further gel shift or change in tryptic digest. Phosphorylation of PDE5 is stimulated by inhibitors, and combination of cGMP and inhibitor does not cause further phosphorylation. Dephosphorylation of PDE5 by either purified phosphoprotein phosphatase-1 or -2A catalytic subunit or by a crude phosphatase mixture is not affected by cGMP or inhibitors, suggesting that phosphorylation itself maintains conformational exposure of the phosphorylation site. The combined results imply that cGMP binding to the catalytic site initiates negative feedback control of many cellular cGMP signaling pathways by directly stimulating phosphorylation and activation of PDE5; by exploiting this molecular mechanism, PDE5 inhibitors stimulate their own potencies.

A 46-amino acid segment in phosphodiesterase-5 GAF-B domain provides for high vardenafil potency over sildenafil and tadalafil and is involved in phosphodiesterase-5 dimerization.

Phosphodiesterase-5 (PDE5) contains a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that contains two mammalian cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlAs (GAFs) (A and B) and a phosphorylation site for cyclic nucleotide-dependent protein kinases (cNPKs). Binding of cGMP to GAF-A increases cNPK phosphorylation of PDE5 and improves catalytic site affinity for cGMP or inhibitors. GAF-B contributes to dimerization of PDE5, inhibition of cGMP binding to GAF-A, and sequestration of the phosphorylation site. To probe potential PDE5 R domain effects on catalytic site affinity for certain inhibitors, four N-terminal truncation mutants were generated: PDE5Delta1-321 contained GAF-B domain, C domain, and the sequence between GAF-A and -B; PDE5Delta1-419 contained GAF-B and C domain; PDE5Delta1-465 contained the C domain and the C-terminal portion of GAF-B; and PDE5Delta1-534 contained only C domain. Truncated proteins with a complete GAF-B were dimers, but those lacking the N-terminal 46 amino acids of GAF-B were monomers, indicating that these residues are vital for GAF-B-mediated PDE5 dimerization. K(m) values of the mutants for cGMP were similar to that of full-length PDE5. All PDE5 constructs had similar affinities for 3-isobutyl-1-methylxanthine, sildenafil, tadalafil, and UK-122764, but mutants containing a complete GAF-B had 7- to 18-fold higher affinity for vardenafil-based compounds compared with those lacking a complete GAF-B. This indicated that the N-terminal 46 amino acids in GAF-B are required for high vardenafil potency. This is the first evidence that PDE5 R domain, and GAF-B in particular, influences affinity and selectivity of the catalytic site for certain classes of inhibitors.

Multiple conformations of phosphodiesterase-5: implications for enzyme function and drug development.

Phosphodiesterase-5 (PDE5) is the target for sildenafil, vardenafil, and tadalafil, which are drugs for treatment of erectile dysfunction and pulmonary hypertension. We report here the crystal structures of a fully active catalytic domain of unliganded PDE5A1 and its complexes with sildenafil or icarisid II. These structures together with the PDE5A1-isobutyl-1-methylxanthine complex show that the H-loop (residues 660-683) at the active site of PDE5A1 has four different conformations and migrates 7-35A upon inhibitor binding. In addition, the conformation of sildenafil reported herein differs significantly from those in the previous structures of chimerically hybridized or almost inactive PDE5. Mutagenesis and kinetic analyses confirm that the H-loop is particularly important for substrate recognition and that invariant Gly(659), which immediately precedes the H-loop, is critical for optimal substrate affinity and catalytic activity.

Phosphodiesterase-5 Gln817 is critical for cGMP, vardenafil, or sildenafil affinity: its orientation impacts cGMP but not cAMP affinity.

The side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other amino acids and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln(775) constrains the orientation of the invariant Gln(817) side chain, which forms bidentate bonds with 5'-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) (Sung, B. J., Hwang, K. Y., Jeon, Y. H., Lee, J. I., Heo, Y. S., Kim, J. H., Moon, J., Yoon, J. M., Hyun, Y. L., Kim, E., Eum, S. J., Park, S. Y., Lee, J. O., Lee, T. G., Ro, S., and Cho, J. M. (2003) Nature 425, 98-102; Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol. Chem. 279, 13095-13101; Zhang, K. Y., Card, G. L., Suzuki, Y., Artis, D. R., Fong, D., Gillette, S., Hsieh, D., Neiman, J., West, B. L., Zhang, C., Milburn, M. V., Kim, S. H., Schlessinger, J., and Bollag, G. (2004) Mol. Cell 15, 279-286). PDE5(Q817A) and PDE5(Q775A) were generated to test the hypotheses that Gln(817) is critical for cyclic nucleotide or inhibitor affinity and that Gln(775) immobilizes the Gln(817) side chain to provide cGMP/cAMP selectivity. Allosteric cGMP binding and the molecular mass of the mutant proteins were unchanged compared with PDE5(WT). For PDE5(Q817A), K(m) for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5(Q775A), K(m) for cGMP was weakened approximately 20-fold but was unchanged for cAMP. For PDE5(Q817A), vardenafil, sildenafil, and IBMX inhibitory potencies were weakened 610-, 48-, and 60-fold, respectively, indicating that Gln(817) is a major determinant of potency, especially for vardenafil, and that binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5(Q775A). It is concluded that Gln(817) is a positive determinant for PDE5 affinity for cGMP and several inhibitors; Gln(775), which perhaps restricts rotation of Gln(817) side chain, is critical for cGMP affinity but has no measurable effect on affinity for cAMP, sildenafil, or vardenafil.