Discovery and modelling studies of natural ingredients from Gaultheria yunnanensis (FRANCH.) against phosphodiesterase-4.

Phosphodiesterase-4 (PDE4) is an anti-inflammatory target for treatment of asthma and chronic obstructive pulmonary disease (COPD). Here, we report the isolation and characterization of 13 compounds (G1-G13) by bioassay-guided fractionation of the ethyl acetate extraction of Gaultheria yunnanensis (FRANCH.), one of which pentacyclic triterpene (G1) has never been reported. Four of them (G1, G2, G4, and G5) inhibit PDE4 with the IC50 values < 20 µM and G1 is the most potent ingredient with an IC50 of 245 nM and moderate selectivity over other PDE families. Molecular dynamics simulations suggest that G1 forms a hydrogen bond with Asn362, in addition to the hydrogen bond with Gln369 and π-π interactions with Phe372, which are commonly observed in the binding of most PDE4 inhibitors. The calculated binding free energies for the interactions of PDE4-G1 and PDE4-G2 are -19.4 and -18.8 kcal/mol, in consistence with the bioassay that G1 and G2 have IC50 of 245 nM and 542 nM, respectively. The modelling results of these active compounds may aid the rational design of novel PDE4 inhibitors as anti-inflammatory agents.

Discovery of a phosphodiesterase 9A inhibitor as a potential hypoglycemic agent.

Phosphodiesterase 9 (PDE9) inhibitors have been studied as potential therapeutics for treatment of diabetes and Alzheimer's disease. Here we report a potent PDE9 inhibitor 3r that has an IC50 of 0.6 nM and >150-fold selectivity over other PDEs. The HepG2 cell-based assay shows that 3r inhibits the mRNA expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase. These activities of 3r, together with the reasonable pharmacokinetic properties and no acute toxicity at 1200 mg/kg dosage, suggest its potential as a hypoglycemic agent. The crystal structure of PDE9-3r reveals significantly different conformation and hydrogen bonding pattern of 3r from those of previously published 28s. Both 3r and 28s form a hydrogen bond with Tyr424, a unique PDE9 residue (except for PDE8), but 3r shows an additional hydrogen bond with Ala452. This structure information might be useful for design of PDE9 inhibitors.

Natural phosphodiesterase-4 (PDE4) inhibitors from Crotalaria ferruginea.

Bioassay-guided fractionation of the ethanol extract of the Chinese folk medicine Crotalaria ferruginea led to the isolation of a new isoflavonoid, 4'-hydroxy-2'-methylalpinum-isoflavone (1), and eight known analogs (2-9). Their structures were elucidated by spectroscopic analysis. Compounds 1, 2, 5, and 8 showed inhibitory activities against phosphodiesterase-4 (PDE4), a therapeutic target of asthma, with IC50 values ranging from 2.57 to 8.94 µM. The possible action mechanism and the structure-activity relationship (SAR) of the active isoflavonoids were explored by using molecular docking and molecular dynamics (MD) simulation methods. Our study herein may explain the anti-inflammatory function of this plant in Chinese folk medicine.

Discovery of 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one as a phosphodiesterase-5 inhibitor and its complex crystal structure.

Phosphodiesterase-5 (PDE5) inhibitors have been approved for the treatment of erectile dysfunction and pulmonary hypertension, but enthusiasm on discovery of PDE5 inhibitors continues for their potential new applications. Reported here is discovery of a series of new PDE5 inhibitors by structure-based design, molecular docking, chemical synthesis, and enzymatic characterization. The best compound, 3-(4-hydroxybenzyl)-1-(thiophen-2-yl)chromeno[2,3-c]pyrrol-9(2H)-one (57), has an IC50 of 17 nM against the PDE5 catalytic domain and good selectivity over other PDE families. The crystal structure of the PDE5 catalytic domain in complex with 57 was determined at 2Å resolution and showed that 57 occupies the same pocket as other PDE5 inhibitors, but has a different binding pattern in detail. On the basis of the binding pattern of 57, a novel scaffold can be proposed as a candidate of PDE inhibitors.

Six new tetraprenylated alkaloids from the South China Sea gorgonian Echinogorgia pseudossapo.

Six new tetraprenylated alkaloids, designated as malonganenones L-Q (1-6), were isolated from the gorgonian Echinogorgia pseudossapo, collected in Daya Bay of Guangdong Province, China. The structures of 1-6 featuring a methyl group at N-3 and a tetraprenyl chain at N-7 in the hypoxanthine core were established by extensive spectroscopic analyses. Compounds 1-6 were tested for their inhibitory activity against the phosphodiesterases (PDEs)-4D, 5A, and 9A, and compounds 1 and 6 exhibited moderate inhibitory activity against PDE4D with IC50 values of 8.5 and 20.3 µM, respectively.

The molecular basis for the selectivity of tadalafil toward phosphodiesterase 5 and 6: a modeling study.

Great attention has been paid to the clinical significance of phosphodiesterase 5 (PDE5) inhibitors, such as sildenafil, tadalafil, and vardenafil widely used for erectile dysfunction. However, sildenafil causes side effects on visual functions since it shows similar potencies to inhibit PDE5 and PDE6, whereas tadalafil gives a high selectivity of 1020-fold against PDE6. Till now, their molecular mechanisms of selectivity of PDE5 versus PDE6 have remained unknown in the absence of the crystal structure of PDE6. In order to elucidate its isoform-selective inhibitory mechanism, a 3D model of PDE6 was constructed by homology modeling, and its interaction patterns with tadalafil plus sildenafil were exploited by molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations. The present work reveals that tadalafil exhibits a less negative predicted binding free energy of -35.21 kcal/mol with PDE6 compared with the value of -41.12 kcal/mol for PDE5, which suggests that tadalafil prefers PDE5 rather than PDE6 and confers a high selectivity for PDE5 versus PDE6. The binding free energy results for tadalafil were consistent with external bioassay studies (IC50 = 5100 and 5 nM toward PDE6 and PDE5, respectively). Two important residues from the Q2 pockets (Val782 and Leu804 in PDE5 and their corresponding Val738 and Met760 in PDE6) were further identified to account for the high selectivity of tadalafil for PDE5 versus PDE6. These findings have shed light on the continuous puzzle of why sildenafil (IC50 = 74 and 6 nM toward PDE6 and PDE5, respectively) causes visual disorders because of its poor selectivity but tadalafil does not. In addition, the homology model of PDE6 can be used to design more potent and selective second-generation PDE5 inhibitors with less inhibitory potency against PDE6.

The molecular basis for the inhibition of phosphodiesterase-4D by three natural resveratrol analogs. Isolation, molecular docking, molecular dynamics simulations, binding free energy, and bioassay.

The phosphodiesterase-4 (PDE4) enzyme is a promising therapeutic target for several diseases. Our previous studies found resveratrol and moracin M to be natural PDE4 inhibitors. In the present study, three natural resveratrol analogs [pterostilbene, (E)-2',3,5',5-tetrahydroxystilbene (THSB), and oxyresveratrol] are structurally related to resveratrol and moracin M, but their inhibition and mechanism against PDE4 are still unclear. A combined method consisting of molecular docking, molecular dynamics (MD) simulations, binding free energy, and bioassay was performed to better understand their inhibitory mechanism. The binding pattern of pterostilbene demonstrates that it involves hydrophobic/aromatic interactions with Phe340 and Phe372, and forms hydrogen bond(s) with His160 and Gln369 in the active site pocket. The present work also reveals that oxyresveratrol and THSB can bind to PDE4D and exhibits less negative predicted binding free energies than pterostilbene, which was qualitatively validated by bioassay (IC50=96.6, 36.1, and 27.0µM, respectively). Additionally, a linear correlation (R(2)=0.953) is achieved for five PDE4D/ligand complexes between the predicted binding free energies and the experimental counterparts approximately estimated from their IC50 values (≈RT ln IC50). Our results imply that hydrophobic/aromatic forces are the primary factors in explaining the mechanism of inhibition by the three products. Results of the study help to understand the inhibitory mechanism of the three natural products, and thus help the discovery of novel PDE4 inhibitors from resveratrol, moracin M, and other natural products.

Identification of novel phosphodiesterase-4D inhibitors prescreened by molecular dynamics-augmented modeling and validated by bioassay.

Phosphodiesterase-4D (PDE4D) has been proved to be a potential therapeutic target against strokes. In the present study, a procedure of integrating pharmacophore, molecular docking, molecular dynamics (MD) simulations, binding free energy calculations, and finally validation with bioassay was developed and described to search for novel PDE4D inhibitors from the SPECS database. Among the 29 compounds selected by our MD-augmented strategy, 15 hits were found with IC50 between 1.9 and 50 µM (a hit rate of 52%) and 6 potent hits showed IC50 less than 10 µM, which suggested that MD simulations can explore the intermolecular interactions of PDE4D-inhibitor complexes more precisely and thus significantly enhanced the hit rate of this screening. The effective and efficient integrated procedures described in this study could be readily applied to screening studies toward other drug targets.

Feeding with supplemental squalene enhances the productive performance in boars.

In the present study, we investigated whether squalene could enhance the reproductive performance of boars. Boars at 12 months of age (weighting at 130+/-5 kg) were randomly allocated into four groups and treated with basal diet (control) or with basal diet supplemental squalene at 10, 20 or 40 mg squalene/kg/day for 60 days. The impact of squalene feeding on the reproductive performance and the levels of serum leptin and testosterone were evaluated before and after feeding. Feeding with squalene at 10 mg/kg/day neither significantly increase the reproductive performance and serum testosterone levels, nor reduced the levels of leptin in boars. Surprisingly, feeding with supplemental squalene at 20 or 40 mg/kg/day significantly improved the reproductive performance as evidenced by dramatically reduced the time for mating, increased semens' volume and motility, and increased the size of litter as compared with that in controls. Furthermore, feeding with a higher dose of squalene significantly reduced the levels of serum leptin, accompanied by elevated levels of testosterone, as compared with that in controls. These data clearly indicate that feeding with squalene can improve the reproductive performance in boars. Therefore, dietary supplementation with squalene may increase the productivity of pig industry and potentially benefit for other animal production.