INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term “facilitated autoinhibition,” whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID. Deacetylation of actin in the CAP/KAc-actin complex activates INF2. Here we use lysine-to-glutamine mutations as acetylmimetics to map the relevant lysines on actin for INF2 regulation, focusing on K50, K61, and K328. Biochemically, K50Q- and K61Q-actin, when bound to CAP2, inhibit full-length INF2 but not INF2 lacking DID. When not bound to CAP, these mutant actins polymerize similarly to WT-actin in the presence or absence of INF2, suggesting that the effect of the mutation is directly on INF2 regulation. In U2OS cells, K50Q- and K61Q-actin inhibit INF2-mediated actin polymerization when expressed at low levels. Direct-binding studies show that the CAP WH2 domain binds INF2-DID with submicromolar affinity but has weak affinity for actin monomers, while INF2-DAD binds CAP/K50Q-actin 5-fold better than CAP/WT-actin. Actin in complex with full-length CAP2 is predominately ATP-bound. These interactions suggest an inhibition model whereby CAP/KAc-actin serves as a bridge between INF2 DID and DAD. In U2OS cells, INF2 is 90-fold and 5-fold less abundant than CAP1 and CAP2, respectively, suggesting that there is sufficient CAP for full INF2 inhibition.

Dengue infection is caused by a mosquito-borne virus, particularly in children, which may even cause death. No effective prevention or therapeutic agents to cure this disease are available up to now. The dengue viral envelope (E) protein was discovered to be a promising target for inhibition in several steps of viral infection. Structure-based virtual screening has become an important technique to identify first hits in a drug screening process, as it is possible to reduce the number of compounds to be assayed, allowing to save resources. In the present study, pharmacophore models were generated using the common hits approach (CHA), starting from trajectories obtained from molecular dynamics (MD) simulations of the E protein complexed with the active inhibitor, flavanone (FN5Y). Subsequently, compounds presented in various drug databases were screened using the LigandScout 4.2 program. The obtained hits were analyzed in more detail by molecular docking, followed by extensive MD simulations of the complexes. The highest-ranked compound from this procedure was then synthesized and tested on its inhibitory efficiency by experimental assays.

Dengue infection is one of the most deleterious public health concerns for two-billion world population being at risk. Plasma leakage, hemorrhage, and shock in severe cases were caused by immunological derangement from secondary heterotypic infection. Flavanone, commonly found in medicinal plants, previously showed potential as anti-dengue inhibitors for its direct antiviral effects and suppressing the pro-inflammatory cytokine from dengue immunopathogenesis. Here, we chemically modified flavanones, pinocembrin and pinostrobin, by halogenation and characterized them as potential dengue 2 inhibitors and performed toxicity tests in human-derived cells and in vivo animal model. Dibromopinocembrin and dibromopinostrobin inhibited dengue serotype 2 at the EC50s of 2.0640 ± 0.7537 and 5.8567 ± 0.5074 µM with at the CC50s of 67.2082 ± 0.9731 and >100 µM, respectively. Both of the compounds also showed minimal toxicity against adult C57BL/6 mice assessed by ALT and Cr levels in day one, three, and eight post-intravenous administration. Computational studies suggested the potential target be likely the NS5 methyltransferase at SAM-binding pocket. Taken together, these two brominated flavanones are potential leads for further drug discovery investigation.

An electrochemical synthesis of 2-aminobenzoxazoles from 2-aminophenols and isothiocyanates was successfully developed in one-pot fashion. Using an inexpensive and widely available NaI and NaCl co-operatively in catalytic amounts, our electrosynthetic...

Cholesterol serves as an essential lipid molecule in various membrane organelles of mammalian cells. The metabolites of cholesterol also play important functions. Acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1), also named as sterol O-acyltransferase 1, is a membrane-bound enzyme residing at the endoplasmic reticulum (ER). It converts cholesterol to cholesteryl esters (CEs) for storage, and is expressed in all cells. CEs cannot partition in membranes; they can only coalesce as cytosolic lipid droplets. Excess CEs are found in the vulnerable region of the brains of patients with late-onset Alzheimer’s disease (AD), and in cell and mouse models for AD. Reducing CE contents by genetic inactivation of ACAT1, or by pharmacological inhibition of ACAT is shown to reduce amyloidopathy and other hallmarks for AD. To account for the various beneficial actions of the ACAT1 blockade (A1B), a working hypothesis is proposed here: the increase in CE contents observed in the AD brain is caused by damages of cholesterol-rich lipid rafts that are known to occur in neurons affected by AD. These damages cause cholesterol to release from lipid rafts and move to the ER where it will be converted to CEs by ACAT1. In addition, the increase in CE contents may also be caused by overloading with cholesterol-rich substances, or through activation of ACAT1 gene expression by various pro-inflammatory agents. Both scenarios may occur in microglia of the chronically inflamed brain. A1B ameliorates AD by diverting the cholesterol pool destined for CE biosynthesis such that it can be utilized more efficiently to repair membrane damage in various organelles, and to exert regulatory actions more effectively to defend against AD. To test the validity of the A1B hypothesis in cell culture and in vivo, the current status of various anti-ACAT1 agents that could be further developed is briefly discussed.

Cholesterol is essential for cellular function and is stored as cholesteryl esters (CEs). CEs biosynthesis is catalyzed by the enzymes acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2), with ACAT1 being the primary isoenzyme in most cells in humans. In Alzheimer’s Disease, CEs accumulate in vulnerable brain regions. Therefore, ACATs may be promising targets for treating AD. F12511 is a high-affinity ACAT1 inhibitor that has passed phase 1 safety tests for antiatherosclerosis. Previously, we developed a nanoparticle system to encapsulate a large concentration of F12511 into a stealth liposome (DSPE-PEG2000 with phosphatidylcholine). Here, we injected the nanoparticle encapsulated F12511 (nanoparticle F) intravenously (IV) in wild-type mice and performed an HPLC/MS/MS analysis and ACAT enzyme activity measurement. The results demonstrated that F12511 was present within the mouse brain after a single IV but did not overaccumulate in the brain or other tissues after repeated IVs. A histological examination showed that F12511 did not cause overt neurological or systemic toxicity. We then showed that a 2-week IV delivery of nanoparticle F to aging 3xTg AD mice ameliorated amyloidopathy, reduced hyperphosphorylated tau and nonphosphorylated tau, and reduced neuroinflammation. This work lays the foundation for nanoparticle F to be used as a possible therapy for AD and other neurodegenerative diseases.

Background: Cholesterol is essential for growth and maintenance of mammalian cells. It is stored as cholesteryl esters by the enzymes acyl-CoA:cholesterol acyltransferases 1 & 2 (ACAT 1 & 2 ) (Sterol O-acyltransferase 1 & 2 ; SOATs in GenBank). ACAT1 blockade (A1B) in macrophages ameliorates various pro-inflammatory responses elicited by lipopolysaccharides (LPS) or by cholesterol loading. In mouse and human brains, Acat1 expression dominates over Acat2 and Acat1 is elevated in many neurodegenerative diseases and in acute neuroinflammation. However, the possible effects of ACAT1 blockade in neuroinflammation, regulated by mediators such as Toll-Like Receptor 4 (TLR4), has not been studied. Methods: We conducted LPS-induced acute neuroinflammation experiments in control vs myeloid specific or neuron specific Acat1 knockout (KO) mice. Furthermore, we evaluated LPS-induced neuroinflammation in the microglial cell line N9 with or without pre-treatment of the small molecule ACAT1-specific inhibitor K-604. Biochemical and microscopy assays were used to monitor inflammatory responses and the fate of TLR4. Results: In vivo studies revealed that Acat1 inactivation in myeloid cell lineage, but not in neurons, markedly attenuated LPS-induced activation of various pro-inflammatory response genes in hippocampus and cortex. Studies in cell culture showed that pre-incubating cells with K-604 significantly ameliorated the pro-inflammatory responses induced by LPS. In cells acutely treated with LPS (for 30 min), pre-incubation with K-604 significantly increased the endocytosis of TLR4, the major transmembrane signaling receptor that mediates LPS-dependent acute neuroinflammation. In cells chronically treated with LPS (for 24-48 hrs), pre-incubation with K-604 significantly decreased the total TLR4 protein content, presumably due to enhanced trafficking of TLR4 to the lysosomes for degradation. For ex vivo evidence, we isolated microglia from adult mice, and found that in mice without LPS stimulation, myeloid Acat1 inactivation altered cellular distribution of TLR4; in mice with LPS stimulation, myeloid Acat1 inactivation decreased the cellular content of TLR4. Conclusion: Blocking ACAT1 in mouse microglia alters the fate of TLR4 and suppresses its ability to participate in pro-inflammatory signaling cascade in response to LPS.

Cholesterol is essential to cellular function and is stored as cholesteryl esters (CEs). CEs biosynthesis is responsible by the enzymes acyl-CoA: cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2), with ACAT1 as the primary isoenzyme in most cells in humans. ACATs are targets for atherosclerosis therapies and may also be promising targets for treating Alzheimer's Disease (AD). F12511 is a high-affinity ACAT1 inhibitor that has passed phase 1 safety tests for anti-atherosclerosis. Previously, we had developed a nanoparticle system to encapsulate a large concentration of F12511 into a stealth liposome (DSPE-PEG2000 with egg phosphatidylcholine). Here, we injected the nanoparticle encapsulated F12511 (nanoparticle F) intravenously (IV) to wild-type (WT) mice and performed HPLC/MS/MS analysis and ACAT enzyme activity measurement. The results demonstrated that F12511 was present within the mouse brain after a single IV but did not over-accumulate in the brain or other tissues after repeated IVs. Histological examination showed that F12511 did not cause overt neurological or systemic toxicity. We then showed that 2-week IV delivery of nanoparticle F to aging 3xTg AD mice ameliorated amyloidopathy, reduced hyperphosphorylated tau and non-phosphorylated tau, and reduced neuroinflammation. This work lays the foundation with nanoparticle F as a possible therapy for AD and other neurodegenerative diseases.