Phosphorylation at Ser289 Enhances the Oligomerization of Tau Repeat R2.

In tauopathies such as Alzheimer's disease (AD), aberrant phosphorylation causes the dissociation of tau proteins from microtubules. The dissociated tau then aggregates into sequent forms from soluble oligomers to paired helical filaments and insoluble neurofibrillary tangles (NFTs). NFTs is a hallmark of AD, while oligomers are found to be the most toxic form of the tau aggregates. Therefore, understanding tau oligomerization with regard to abnormal phosphorylation is important for the therapeutic development of AD. In this study, we investigated the impact of phosphorylated Ser289, one of the 40 aberrant phosphorylation sites of full-length tau proteins, on monomeric and dimeric structures of tau repeat R2 peptides. We carried out intensive replica exchange molecular dynamics simulation with a total simulation time of up to 0.1 ms. Our result showed that the phosphorylation significantly affected the structures of both the monomer and the dimer. For the monomer, the phosphorylation enhanced ordered-disordered structural transition and intramolecular interaction, leading to more compactness of the phosphorylated R2 compared to the wild-type one. As to the dimer, the phosphorylation increased intermolecular interaction and ß-sheet formation, which can accelerate the oligomerization of R2 peptides. This result suggests that the phosphorylation at Ser289 is likely to promote tau aggregation. We also observed a phosphorylated Ser289-Na+-phosphorylated Ser289 bridge in the phosphorylated R2 dimer, suggesting an important role of cation ions in tau aggregation. Our findings suggest that phosphorylation at Ser289 should be taken into account in the inhibitor screening of tau oligomerization.

In silico binding affinity prediction for metabotropic glutamate receptors using both endpoint free energy methods and a machine learning-based scoring function.

Metabotropic glutamate receptors (mGluRs) play an important role in regulating glutamate signal pathways, which are involved in neuropathy and periphery homeostasis. mGluR4, which belongs to Group III mGluRs, is most widely distributed in the periphery among all the mGluRs. It has been proved that the regulation of this receptor is involved in diabetes, colorectal carcinoma and many other diseases. However, the application of structure-based drug design to identify small molecules to regulate the mGluR4 receptor is limited due to the absence of a resolved mGluR4 protein structure. In this work, we first built a homology model of mGluR4 based on a crystal structure of mGluR8, and then conducted hierarchical virtual screening (HVS) to identify possible active ligands for mGluR4. The HVS protocol consists of three hierarchical filters including Glide docking, molecular dynamic (MD) simulation and binding free energy calculation. We successfully prioritized active ligands of mGluR4 from a set of screening compounds using HVS. The predicted active ligands based on binding affinities can almost cover all the experiment-determined active ligands, with only one ligand missed. The correlation between the measured and predicted binding affinities is significantly improved for the MM-PB/GBSA-WSAS methods compared to the Glide docking method. More importantly, we have identified hotspots for ligand binding, and we found that SER157 and GLY158 tend to contribute to the selectivity of mGluR4 ligands, while ALA154 and ALA155 could account for the ligand selectivity to mGluR8. We also recognized other 5 key residues that are critical for ligand potency. The difference of the binding profiles between mGluR4 and mGluR8 can guide us to develop more potent and selective modulators. Moreover, we evaluated the performance of IPSF, a novel type of scoring function trained by a machine learning algorithm on residue-ligand interaction profiles, in guiding drug lead optimization. The cross-validation root-mean-square errors (RMSEs) are much smaller than those by the endpoint methods, and the correlation coefficients are comparable to the best endpoint methods for both mGluRs. Thus, machine learning-based IPSF can be applied to guide lead optimization, albeit the total number of actives/inactives are not big, a typical scenario in drug discovery projects.

Anti-inflammatory effects of Aureusidin in LPS-stimulated RAW264.7 macrophages via suppressing NF-κB and activating ROS- and MAPKs-dependent Nrf2/HO-1 signaling pathways.

Aureusidin, a naturally-occurring flavonoid, is found in various plants of Cyperaceae such as Heleocharis dulcis (Burm. f.) Trin., but its pharmacological effect and active mechanism are rarely reported. This study aimed to investigate the anti-inflammatory effect and action mechanism of Aureusidin in LPS-induced mouse macrophage RAW264.7 cells. The results suggested that lipopolysaccharide (LPS)-induced nitric oxide (NO), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) production were obviously inhibited by Aureusidin. Moreover, Aureusidin also significantly decreased the mRNA expression of various inflammatory factors in LPS-stimulated RAW264.7 cells. Furthermore, mechanistic studies showed that Aureusidin significantly inhibited nuclear transfer of nuclear factor-κB (NF-κB), while increasing the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) as well as expression of Nrf2 target genes such as heme oxygenase (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1), but the addition of the HO-1 inhibitor Sn-protoporphyrin (Snpp) significantly abolished the anti-inflammatory effect of Aureusidin in LPS-stimulated RAW264.7 cells, confirming the view that HO-1 was involved in the anti-inflammatory effect. In addition, Aureusidin increased the levels of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) phosphorylation in RAW264.7 cells. Antioxidant N-acetylcysteine (NAC) or three MAPK inhibitors blocked the nuclear translocation of Nrf2 and HO-1 expression induced by Aureusidin, indicating that Aureusidin activated the Nrf2/HO-1 signaling pathway through ROS and MAPKs pathways. At the same time, co-treatment with the NAC blocked the phosphorylation of MAPKs. Results from molecular docking indicated that Aureusidin inhibited the NF-κB pathway by covalently binding to NF-κB. Thus, Aureusidin exerted the anti-inflammatory activity through blocking the NF-κB signaling pathways and activating the MAPKs and Nrf2/HO-1 signaling pathways. Based on the above results, Aureusidin may be an attractive therapeutic candidate for the inflammation-related diseases.