Tissue microRNA-182 expression level and its potential prognostic value for papillary thyroid carcinoma.

BACKGROUND: In previous study, qRT-PCR analysis revealed significantly higher miR-182 levels in papillary thyroid carcinoma (PTC) than matched normal tissues. However, the clinical significance and prognostic value of miR-182 have not been investigated in PTC until now. METHODS: 151 pairs of PTC and adjacent normal thyroid tissues were obtained from Affiliated Hospital of Weifang Medical University from February 2008 to January 2015. The Chi square test was used to analyze the relationship between miR-182 expression and the clinicopathological characteristics. We used the Kaplan-Meier method and the log-rank test in univariate survival analysis, and we used the Cox proportional hazards regression model in our multivariate analysis. RESULTS: The relative expression of miR-182 in PTC samples was significantly higher than that of matched normal tissues (P<0.001). The high expression level of tissue miR-182 was statistically correlated with extrathyroidal invasion (P=0.009), cervical lymphnode metastasis (P=0.015), and TNM staging (P=0.001). The Kaplan-Meier method revealed that higher miR-182 expression level was correlated with significantly reduced overall survival. Furthermore, multivariate survival analysis revealed that miR-182 expression level (HR=2.882, 95% CI: 1.289-10.928, P=0.013) was significantly correlated with the poor prognosis of PTC patients. CONCLUSIONS: Overexpression of miR-182 is associated with aggressive clinicopathologic characteristics of PTC, and miR-182 might be a novel prognostic molecular marker of PTC.

Discovery of Novel Phosphodiesterase-2A Inhibitors by Structure-Based Virtual Screening, Structural Optimization, and Bioassay.

Phosphodiesterase-2A (PDE2A) is a potential therapeutic target for treatment of Alzheimer's disease and pulmonary hypertension. However, most of the current PDE2A inhibitors have moderate selectivity over other PDEs. In the present study, we described the discovery of novel PDE2A inhibitors by structure-based virtual screening combining pharmacophore model screening, molecular docking, molecular dynamics simulations, and bioassay validation. Nine hits out of 30 molecules from the SPECS database (a hit rate of 30%) inhibited PDE2A with affinity less than 50 µM. Optimization of compound AQ-390/10779040 (IC50 = 4.6 µM) from the virtual screening, which holds a novel scaffold of benzo[cd]indol-2(1H)-one among PDE inhibitors, leads to discovery of a new compound LHB-8 with a significant improvement of inhibition (IC50 = 570 nM). The modeling studies demonstrated that LHB-8 formed an extra hydrogen bond with Asp808 and a hydrophobic interaction with Thr768, in addition to the common interactions with Gln859 and Phe862 of PDE2A. The novel scaffolds discovered in the present study can be used for rational design of PDE2A inhibitors with high affinity.

-374T/A polymorphism of the receptor for advanced glycation end products is associated with decreased risk of breast cancer in a Chinese population.

PURPOSE: we aimed to investigate the receptor for advanced glycation end products (RAGE) -374T/A polymorphism and breast cancer risk in a Chinese population. METHODS: The study subjects included 188 women with histologically confirmed breast cancer and 210 controls. The RAGE genotypes were determined using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay. Pearson's χ(2) test was used to test the association between cases and controls and genotype frequencies. The association between the polymorphism and risk of breast cancer was estimated by odds ratio (OR) and 95% confidence interval (95% CI). RESULTS: The AA genotype was significantly higher in breast cancer patients than in controls (37.77% vs. 28.10%, P = 0.002). Furthermore, the A allele frequency was significantly higher in the case group than in the control group (55.32% vs. 42.14%, P < 0.001). With the TT genotype as reference, the adjusted OR for AA homozygous carriers reached to 0.36 (95% CI: 0.17-0.88; P = 0.03). Under the dominant model of inheritance, the TA+AA genotype was associated with significantly decreased risk for breast cancer (adjusted OR = 0.38, 95% CI = 0.27-0.87; P = 0.02). The A allele carriage also presented a lower risk for breast cancer (adjusted OR = 0.42; 95% CI, 0.33-0.91; P = 0.04). CONCLUSION: Our findings suggest that the polymorphic variants of RAGE-374T/A may have an influence on breast cancer risk among Chinese women.

Molecular dynamics-based discovery of novel phosphodiesterase-9A inhibitors with non-pyrazolopyrimidinone scaffolds.

Phosphodiesterase-9A (PDE9A) is a promising therapeutic target for the treatment of diabetes and Alzheimer's disease (AD). The Pfizer PDE9A inhibitor PF-04447943 has completed Phase II clinical trials in subjects with mild to moderate AD in 2013. However, most of the reported PDE9A inhibitors share the same scaffold as pyrazolopyrimidinone, which lacks structural diversity and is unfavorable for the development of novel PDE9A inhibitors. In the present study, a combinatorial method including pharmacophores, molecular docking, molecular dynamics simulations, binding free energy calculations, and bioassay was used to discover novel PDE9A inhibitors with new scaffolds rather than pyrazolopyrimidinones from the SPECS database containing about 200,000 compounds. As a result, 15 hits out of 29 molecules (a hit rate of 52%) with five novel scaffolds were identified to be PDE9A inhibitors with inhibitory affinities no more than 50 µM to enrich the structural diversity, different from the pyrazolopyrimidinone-derived family. The high hit ratio of 52% for this virtual screening method indicated that the combinatorial method is a good compromise between computational cost and accuracy. Binding pattern analyses indicate that those hits with non-pyrazolopyrimidinone scaffolds can bind the same active site pocket of PDE9A as classical PDE9A inhibitors. In addition, structural modification of compound AG-690/40135604 (IC50=8.0 µM) led to a new one, 16, with an improved inhibitory affinity of 2.1 µM as expected. The five novel scaffolds discovered in the present study can be used for the rational design of PDE9A inhibitors with higher affinities.

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.

Palladium-catalyzed oxidative C-H bond acylation of N-nitrosoanilines with toluene derivatives: a traceless approach to synthesize N-alkyl-2-aminobenzophenones.

A palladium-catalyzed cascade cross-coupling of N-nitroso-anilines and toluene derivatives for the direct synthesis of N-alkyl-2-aminobenzophenones is described. N-nitroso groups in anilines can act as the traceless directing groups while toluene derivatives can serve as effective acyl precursors under mild reaction conditions.

Ab Initio QM/MM Study Shows a Highly Dissociated SN2 Hydrolysis Mechanism for the cGMP-Specific Phosphodiesterase-5.

Phosphodiesterases (PDEs) are the sole enzymes hydrolyzing the important second messengers cGMP and cAMP and have been identified as therapeutic targets for several diseases. The most successful examples are PDE5 inhibitors (i.e., sildenafil and tadalafil), which have been approved for the treatment of male erectile dysfunction and pulmonary hypertension. However, the side effects mostly due to nonselective inhibition toward other PDE isoforms, set back the clinical usage of PDE5 inhibitors. Until now, the exact catalytic mechanism of the substrate cGMP by PDE5 is still unclear. Herein, the first computational study on the catalytic hydrolysis mechanism of cGMP for PDE5 (catalytic domain) is performed by employing the state-of-the-art ab initio quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations. Our simulations show a SN2 type reaction procedure via a highly dissociated transition state with a reaction barrier of 8.88 kcal/mol, which is quite different from the previously suggested hydrolysis mechanism of cAMP for PDE4. Furthermore, the subsequent ligand exchange and the release of the product GMP have also been investigated by binding energy analysis and MD simulations. It is deduced that ligand exchange would be the rate-determining step of the whole reaction, which is consistent with many previous experimental results. The obtained mechanistic insights should be valuable for not only the rational design of more specific inhibitors toward PDE5 but also understanding the general hydrolysis mechanism of cGMP-specific PDEs.

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.