Noncanonical autophagy is a new strategy to inhibit HSV-1 through STING1 activation

STING1 (stimulator of interferon response cGAMP interactor 1) plays an essential role in immune responses for virus inhibition via inducing the production of type I interferon, inflammatory factors and macroautophagy/autophagy. In this study, we found that STING1 activation could induce not only canonical autophagy but also non-canonical autophagy (NCA) which is independent of the ULK1 or BECN1 complexes to form MAP1LC3/LC3-positive structures. Whether STING1-induced NCA has similar characters and physiological functions to canonical autophagy is totally unknown. Different from canonical autophagy, NCA could increase single-membrane structures and failed to degrade long-lived proteins, and could be strongly suppressed by interrupting vacuolar-type H+-translocating ATPase (V-ATPase) activity. Importantly, STING1-induced NCA could effectively inhibit DNA virus HSV-1 in cell model. Moreover, STING1[1-340], a STING1 mutant lacking immunity and inflammatory response due to deletion of the tail end of STING1, could degrade virus through NCA alone, suggesting that the antiviral effect of activated STING1 could be separately mediated by inherent immunity, canonical autophagy, and NCA. In addition, the translocation and dimerization of STING1 do not rely on its immunity function and autophagy pathway. Similar to canonical autophagy, LC3-positive structures of NCA induced by STING1 could finally fuse with lysosomes, and the degradation of HSV-1 could be reverted by inhibition of lysosome function, suggesting that the elimination of DNA virus via NCA still requires the lysosome pathway. Collectively, we proved that besides its classical immunity function and canonical autophagy pathway, STING1-induced NCA is also an efficient antiviral pathway for the host cell.</p

S1P2-siRNA had no effect on the activation of NF-κB pathway induced by HG in GMCs.

<p>GMCs were treated with HG under S1P2-siRNA pretreatment condition, The protein levels of S1P2 receptor (A), p65 protein contents in nuclear (B), cytoplasm (C) and total p65 expression (D) were analyzed by Western Blot assay in GMCs. (E) DNA binding activity of NF-<b>κ</b>B was determined by EMSA. Data are represented as Means ± SDs, *<i>P</i><0.05 vs. control.</p

BBR and PDTC suppressed S1P2 receptor and FN expression in GMCs under HG condition.

<p>GMCs were treated with BBR (10, 30, 90 µM) and PDTC (100 µM) for 24h, protein bands (A) were detected using the enhanced chemiluminescence detection system, S1P2 receptor (B) and FN (C) expression were analyzed by Western Blot assay. Data are represented as Means ± SDs, *<i>P</i><0.05 vs. control, #<i>P</i><0.05 vs. HG.</p

Effects of BBR on S1P2 receptor expression in GMCs under HG condition.

<p>GMCs were treated with different dose of BBR under HG condition. After 24 h of incubation, the levels of mRNA (A) and protein (B) of S1P2 receptor were evaluated using real-time PCR and Western blot, respectively. Expression (C) and distribution (D) of S1P2 receptor on the membrane of GMCs was detected by Western blot and LSCM. Data are represented as Means ± SDs, *<i>P</i><0.05 vs. normal glucose (NG), #<i>P</i><0.05 vs. HG.</p

Schematic representation of the possible mechanism of BBR on the S1P2 receptor.

<p>Subjecting GMCs to HG results in the activation of NF-<b>κ</b>B signaling pathway, and thus up-regulates S1P2 receptor expression, which contributes to the increased FN levels. In contrast, pretreatment with BBR inhibits S1P2 receptor and FN levels possibly by down-regulating NF-<b>κ</b>B activation.</p

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

AMDE-1 is a potent cytotoxic agent.

<p>(<b>A-B</b>) HeLa cells were treated with AMDE (A) or CQ (B) at different doses for 48 hours. Cell death was measured. The dose responses were fitted with a logarithmic regression (A) or a second order polynomial regression (B) algorithm, respectively. (<b>C)</b> Cells were treated with CQ (50 μM) or AMDE (2.5 μM) for 48 hours. Cell death was then measured. (<b>D</b>) HCT116 and CCD-18Co cells were treated with AMDE at different concentrations for 48 hours. Cell death was measured. The dose response was fitted with a logarithmic regression algorithm. (<b>E</b>) HCT16 was treated with or without AMDE-1 (2.5 μM), zVAD (20 μM), and/or necrostatin-1 (necro, 40 μM) for 48 hours. Cell death was measured. Values represent means ± SD from three independent experiments. *: p<0.05, ***: p = 0.001.</p

BBR inhibited NF-κB activation induced by HG in GMCs.

<p>GMCs were treated with BBR (10, 30, 90 µM) and PDTC (100 µM) for 24 h. Protein bands (A) were detected using the enhanced chemiluminescence detection system, p65 protein contents in nuclear (B), cytoplasm (C) and total p65 expression (D) were analyzed by Western Blot assay. (E) DNA binding activities of NF-<b>κ</b>B were determined by EMSA. Data are represented as Means ± SDs, *<i>P</i><0.05 vs. control, #<i>P</i><0.05 vs. HG.</p

BBR reduced mRNA and protein expression of S1P2 receptor in diabetic rat kidneys.

<p>STZ-induced diabetic rats were treated for 12 weeks with BBR, and the levels of mRNA (A) and protein (B) of S1P2 receptor were evaluated by real-time PCR and Western blot, respectively. Data are represented as Means ± SDs, *<i>P</i><0.05 vs. control, #<i>P</i><0.05 vs. diabetes.</p

BBR reduced FN expression in diabetic rat kidneys.

<p>STZ-induced diabetic rats were treated for 12 weeks with BBR, and the levels of FN were detected by Western blot assay. Data are represented as Means ± SDs, *<i>P</i><0.05 vs. control, #<i>P</i><0.05 vs. diabetes.</p