ABSTRACT
The oxidative metabolism of tocopherols and tocotrienols by monooxygenases is a key factor in the plasma and tissue clearance of forms of vitamin E other than alpha-tocopherol. It is well known that a commonly ingested form of vitamin E, gamma-tocopherol, has greatly reduced plasma half-life (faster clearance) than alpha-tocopherol. The tocotrienols are metabolized even faster than gamma-tocopherol. Both gamma-tocopherol and alpha- and delta-tocotrienol possess intriguing biological activities that are different from alpha-tocopherol, making them potentially of interest for therapeutic use. Unfortunately, the fast clearance of non-alpha-tocopherols from animal tissues is a significant hurdle to maximizing their effect(s) as dietary supplements. We report here the design and synthesis of N-heterocycle-containing analogues of alpha-tocopherol that act as inhibitors of Cyp4F2, the key monooxygenase responsible for omega-hydroxylation of the side chain of tocols. In particular, an omega-imidazole containing compound, 1, [(R)-2-(9-(1H-imidazol-1-yl)nonyl)-2,5,7,8-tetramethylchroman-6-ol] had an ED(50) for inhibition of gamma-CEHC production from gamma-tocopherol of approximately 1 nM when tested in HepG2 cells in culture. Furthermore, feeding of 1 to mice along with rapidly metabolized delta-tocopherol, resulted in a doubling of the delta-tocopherol/alpha-tocopherol ratio in liver (P<0.05). Thus, 1 may be a useful adjuvant to the therapeutic use of non-alpha-tocopherols.
Subject(s)
Benzimidazoles/chemical synthesis , Benzimidazoles/pharmacology , Tocopherols/antagonists & inhibitors , Tocopherols/metabolism , Triazoles/chemical synthesis , Vitamin E/analogs & derivatives , Animals , Benzimidazoles/chemistry , Bile/enzymology , Cell Line , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Humans , Liver/enzymology , Magnetic Resonance Spectroscopy , Mass Spectrometry , Mice , Mixed Function Oxygenases/antagonists & inhibitors , Mixed Function Oxygenases/metabolism , Tocopherols/blood , Tocotrienols/metabolism , Triazoles/chemistry , Triazoles/pharmacologyABSTRACT
The Ca(2+)-triggered merger of two apposed membranes is the defining step of regulated exocytosis. CHOL is required at critical levels in secretory vesicle membranes to enable efficient, native membrane fusion: CHOL-sphingomyelin enriched microdomains organize the site and regulate fusion efficiency, and CHOL directly supports the capacity for membrane merger by virtue of its negative spontaneous curvature. Specific, structurally dissimilar lipids substitute for CHOL in supporting the ability of vesicles to fuse: diacylglycerol, alphaT, and phosphatidylethanolamine support triggered fusion in CHOL-depleted vesicles, and this correlates quantitatively with the amount of curvature each imparts to the membrane. Lipids of lesser negative curvature than cholesterol do not support fusion. The fundamental mechanism of regulated bilayer merger requires not only a defined amount of membrane-negative curvature, but this curvature must be provided by molecules having a specific, critical spontaneous curvature. Such a local lipid composition is energetically favorable, ensuring the necessary "spontaneous" lipid rearrangements that must occur during native membrane fusion-Ca(2+)-triggered fusion pore formation and expansion. Thus, different fusion sites or vesicle types can use specific alternate lipidic components, or combinations thereof, to facilitate and modulate the fusion pore.
Subject(s)
Calcium/chemistry , Membrane Fluidity , Membrane Fusion , Models, Chemical , Phospholipids/chemistry , Computer Simulation , Surface PropertiesABSTRACT
Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin alpha-tocopherol were prepared incorporating fluorophores at the terminus of omega-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of alpha-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein's binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (alpha-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2R,4'R,8'R-alpha-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of alpha-TTP-mediated inter-membrane transfer of alpha-tocopherol and to investigate the mechanism of alpha-TTP function at membranes of different composition.
Subject(s)
Carrier Proteins/drug effects , Tocopherols/chemical synthesis , Tocopherols/pharmacokinetics , Binding, Competitive/drug effects , Fluorescence , Humans , Models, Molecular , Molecular Structure , Protein Binding/drug effects , Recombinant Proteins/drug effects , Structure-Activity Relationship , Tocopherols/chemistry , X-Ray DiffractionABSTRACT
Novel fluorescent analogues of alpha-tocopherol have been prepared that incorporate the useful fluorophores nitrobenoxadiazyl (NBD) and anthroyloxy (AO). Both fluorescent tocopherol analogues bind specifically to recombinant human tocopherol transfer protein (hTTP). The NBD-alpha-tocopherol is particularly useful for protein-binding assays, whereas the AO-alpha-tocopherol was designed to be one of a pair of chromophores for a fluorescence resonance energy transfer (FRET) assay of intervesicular tocopherol transfer. It is now possible to follow AO-alpha-tocopherol transfer from donor lipid vesicles composed of predominantly phosphatidylcholine (PC) to acceptor lipid vesicles containing PC and a quenching lipid NBD-PE (2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[7-nitro-2-1,3-benzoxadiazol-4-yl]). The presence of hTTP substantially increases the rate of AO-alpha-tocopherol transfer over the uncatalyzed spontaneous rate.