Targeting Caveolin-1 and Claudin-5 with AY9944, Improve Blood-Brain Barrier Permeability; Computational Simulation and Experimental Study.

The current study aimed to determine the protective effect of AY9944 related to Caveolin-1 and Claudin-5 role in lipid raft, which can rescue the blood-brain barrier from enhanced permeability. Therefore, in vivo analyses were performed following ischemia in normal, ischemic, and AY9944-treated animal groups. The results revealed that AY9944 reduced the infarct size, edema, and brain water content. The extravasation of Alb-Alexa 594 and biocytin-TMR was minimum in the AY9944-treated animals. The results showed a significant decrease in the expression level of Caveolin-1 over 8 h and 48 h and a remarkable increase in the level of Claudin-5 over 48 h following ischemia in AY9944-treated animals. Molecular docking simulation demonstrated that AY9944 exerts a possible protective role via attenuating the interaction of the Caveolin-1 and cholesterol in lipid raft. These findings point out that AY9944 plays a protective role in stroke by means of blood-brain barrier preservation. Proper neural function essentially needs a constant homeostatic brain environment which is provided by the blood-brain barrier. Rescuing blood-brain barrier from enhanced permeability via inducing the protective effect of AY9944 related to caveolin-1 and claudin-5 role in lipid raft was the aim of the current study.

Identification of Environmental Quaternary Ammonium Compounds as Direct Inhibitors of Cholesterol Biosynthesis.

In this study, we aim to identify environmental molecules that can inhibit cholesterol biosynthesis, potentially leading to the same biochemical defects as observed in cholesterol biosynthesis disorders, which are often characterized by congenital malformations and developmental delay. Using the Distributed Structure-Searchable Toxicity (DSSTox) Database Network developed by EPA, we first carried out in silico screening of environmental molecules that display structures similar to AY9944, a known potent inhibitor of 3ß-hydroxysterol-Δ(7)-reductase (DHCR7)-the last step of cholesterol biosynthesis. Molecules that display high similarity to AY9944 were subjected to test in mouse and human neuroblastoma cells for their effectiveness in inhibiting cholesterol biosynthesis by analyzing cholesterol and its precursor using gas chromatography-mass spectrometry. We found that a common disinfectant mixture, benzalkonium chlorides (BACs), exhibits high potency in inhibiting DHCR7, as suggested by greatly elevated levels of the cholesterol precursor, 7-dehydrocholesterol (7-DHC). Subsequent structure-activity studies suggested that the potency of BACs as Dhcr7 inhibitors decrease with the length of their hydrocarbon chain: C10 > C12 ≫ C14 > C16. Real-time qPCR analysis revealed upregulation of the genes related to cholesterol biosynthesis and downregulation of the genes related to cholesterol efflux, suggesting a feedback response to the inhibition. Furthermore, an oxidative metabolite of 7-DHC that was previously identified as a biomarker in vivo was also found in cells exposed to BACs by liquid chromatography-mass spectrometry. Our findings suggest that certain environmental molecules could potently inhibit cholesterol biosynthesis, which could be a new link between environment and developmental disorders.

High affinity of sigma 1-binding sites for sterol isomerization inhibitors: evidence for a pharmacological relationship with the yeast sterol C8-C7 isomerase.

1. The sigma-drug binding site of guinea-pig liver is carried by a protein which shares significant amino acid sequence similarities with the yeast sterol C8-C7 isomerase (ERG2 protein). Pharmacologically-but not structurally-the sigma 1-site is also related to the emopamil binding protein, the mammalian sterol C8-C7 isomerase. We therefore investigated if sterol C8-C7 isomerase inhibitors are high affinity ligands for the (+)-[3H]-pentazocine labelled sigma 1-binding site. 2. Among the compounds which bound with high affinity to native hepatic and cerebral as well as to yeast expressed sigma 1-binding sites were the agricultural fungicide fenpropimorph (Ki 0.005 nM), the antihypocholesterinaemic drugs triparanol (Ki 7.0 nM), AY-9944 (Ki, 0.46 nM) and MDL28,815 (Ki 0.16 nM), the enantiomers of the ovulation inducer clomiphene (Ki 5.5 and 12 nM, respectively) and the antioestrogene tamoxifen (Ki 26 nM). 3. Except for tamoxifen these affinities are essentially identical with those for the [3H]-ifenprodil labelled sterol C8-C7 isomerase of S. cerevisiae. This demonstrates that sigma 1-binding protein and yeast isomerase are not only structurally but also pharmacologically related. Because of its affiliations with yeast and mammalian sterol isomerases we propose that the sigma 1-binding site is localized on a sterol isomerase related protein, involved in postsqualene sterol biosynthesis.

Evolución de las inclusiones citoplasmáticas inducidas por Y-9944 en nervio periférico: análisis cuantitativo / Evolution of the cytoplasmic inclusions induced for AY-9944 in peripheral nerve: quantitative analysis

En el presente trabajo se estudia la evolución morfológica de inclusiones intracitoplasmáticas en ratas lactantes tratadas con AY-9944 (trans-1, 4-bis (2-clorobencilaminometil ciclohexano dihidroclórido), un inhibidor de la biosíntesis de colesterol. Para ello, ratas lactantes fueron inyectadas intraperitonealmente a los tres dias de edad con una dosis única de AY-9944, y fueron sacrificadas juntas con sus correspondientes controles a los 5,10 minutos, 1 y 8 dias de inyectadas. El análisis se basó en la cuantificación, a nivel de microscopía electrónica, del número de inclusiones totales, y del número de inclusiones de cada tipo morfológico que aparecían en cada uno de los tiempos estudiados. Los resultados han indicado: primero, un aumento progresivo del número total de inclusiones desde los 5 minutos hasta los 8 dias de inyectado el inhibidor; y segundo, una variación en los tipos morfológicos de inclusiones predominantes en los tiempos estudiados, en los animales inyectados; en comparación con la ausencia de inclusiones en los respectivos controles