RESUMEN
Arylation via ortho C-H activation by the aid of directing groups has been explored recently by many researchers. Herein, a palladium-catalyzed C-H arylation using 8-aminoquinoline as a bidentate directing group has been developed. The reaction furnishes only C-H arylation, unlike previous methods where cyclization to corresponding isoquinolones is observed. More interestingly, sequential C-H functionalization was observed when methylacrylate and acrylonitrile was added; this led to C-H olefination with the aryl group, which was installed from the aryne precursor.
Asunto(s)
Paladio , CatálisisRESUMEN
Cholesterol is a key steroidal, lipid biomolecule found abundantly in plasma membranes of eukaryotic cells. It is an important structural component of cellular membranes and regulates membrane fluidity and permeability. Cholesterol is also essential for normal functioning of key proteins including ion-channels, G protein-coupled receptors (GPCRs), membrane bound steroid receptors, and receptor kinases. It is thought that cholesterol exerts its actions via specific binding to chiral proteins and lipids as well as through non-specific physiochemical interactions. Distinguishing between the specific and the non-specific interactions can be difficult. Although much remains unclear, progress has been made in recent years by utilizing ent-cholesterol, the enantiomer of natural cholesterol (nat-cholesterol) as a probe. Ent-Cholesterol is the non-superimposable mirror image of nat-cholesterol and exhibits identical physiochemical properties as nat-cholesterol. Hence, if the biological effects of cholesterol result solely due to membrane effects, it is expected that there will be no difference between ent-cholesterol and nat-cholesterol. However, when direct binding with chiral proteins and lipids is involved, the enantiomer is expected to potentially elicit significantly different, measurable effects due to formation of diastereomeric complexes. In this chapter, we have reviewed the literature related to ent-cholesterol and its use as a probe for various biophysical and biological interactions of cholesterol.
Asunto(s)
Membrana Celular/química , Colesterol/química , Proteínas de la Membrana/química , Lípidos de la Membrana/química , EstereoisomerismoRESUMEN
Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo) by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine-rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.
Asunto(s)
Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Esteroles/metabolismo , Aminoácidos/metabolismo , Animales , Sitios de Unión , Secuencia Conservada , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Ligandos , Ratones , Modelos Moleculares , Células 3T3 NIH , Receptores Patched , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptor Smoothened , Esteroles/química , Relación Estructura-Actividad , Proteínas Wnt/metabolismoRESUMEN
How cholesterol is sensed to maintain homeostasis has been explained by direct binding to a specific protein, Scap, or through altering the physical properties of the membrane. The enantiomer of cholesterol (ent-cholesterol) is a valuable tool in distinguishing between these two models because it shares nonspecific membrane effects with native cholesterol (nat-cholesterol), but not specific binding interactions. This is the first study to compare ent- and nat-cholesterol directly on major molecular parameters of cholesterol homeostasis. We found that ent-cholesterol suppressed activation of the master transcriptional regulator of cholesterol metabolism, SREBP-2, almost as effectively as nat-cholesterol. Importantly, ent-cholesterol induced a conformational change in the cholesterol-sensing protein Scap in isolated membranes in vitro, even when steps were taken to eliminate potential confounding effects from endogenous cholesterol. Ent-cholesterol also accelerated proteasomal degradation of the key cholesterol biosynthetic enzyme, squalene monooxygenase. Together, these findings provide compelling evidence that cholesterol maintains its own homeostasis not only via direct protein interactions, but also by altering membrane properties.
Asunto(s)
Colesterol/química , Colesterol/metabolismo , Animales , Células CHO , Membrana Celular/metabolismo , Cromatografía en Capa Delgada/métodos , Cricetinae , Homeostasis , Metabolismo de los Lípidos , Lípidos/química , Unión Proteica , Escualeno-Monooxigenasa/química , Estereoisomerismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Tripsina/químicaRESUMEN
The activity (Po) of large-conductance voltage/Ca(2+)-gated K(+) (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel-forming α (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol's mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the ß configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol≥coprostanol≥cholestanol>>>epicholesterol) did not follow molecular area rank (coprostanol>>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of α homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK α subunit itself.
Asunto(s)
Colesterol/análogos & derivados , Colesterol/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Esteroles/metabolismo , Animales , Membrana Celular/metabolismo , Membrana Celular/fisiología , Colesterol/química , Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Conformación Molecular , Dominios y Motivos de Interacción de Proteínas , Ratas , Esteroles/químicaRESUMEN
ent-Cholesterol was synthesized in 16 steps from commercially available (S)-citronellol. The overall yield for the synthesis was 2.0%. This route is amenable to gram-scale preparation of ent-cholesterol. Isotopic incorporation near the end of the synthesis was achieved using labeled methyl iodide. This synthesis is the most practical to date and will make ent-cholesterol more readily available to use as a probe of the function and metabolism of cholesterol.
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Colesterol/síntesis química , Monoterpenos/química , Monoterpenos Acíclicos , Colesterol/química , Conformación Molecular , EstereoisomerismoRESUMEN
The alpha, zeta, and epsilon isoforms of diacylglycerol kinase exhibit a high degree of stereospecificity in the phosphorylation of diacylglycerol. In comparison, a multiple lipid kinase, MuLK, shows much less stereospecificity, phosphorylating 1,2-dioleoylglycerol only approximately 2-3 times more rapidly than 2,3-dioleoylglycerol. The alpha and zeta isoforms of diacylglycerol kinase are inhibited by 2,3-dioleoylglycerol, but not the more substrate-selective epsilon isoform. The inhibition by 2,3-dioleoylglycerol is uncompetitive. This corresponds to a kinetic scheme in which the inhibitor can bind to the enzyme-substrate complex, but not to the free enzyme. Our data indicate that despite their similar structures, 1,2-dioleoylglycerol and 2,3-dioleoylglycerol do not compete for the active site of these three isoforms of diacylglycerol kinase. We suggest that the 2,3-dioleoylglycerol binds to a site on the alpha and zeta isoforms of diacylglycerol kinase that is exposed as a consequence of the substrate binding to the active site. The chiral specificity of these enzymes thus mimics the substrate specificity, with MuLK being the least selective and the epsilon isoform of diacylglycerol kinase exhibiting the greatest selectivity.
Asunto(s)
Diacilglicerol Quinasa/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Sitios de Unión , Diacilglicerol Quinasa/antagonistas & inhibidores , Diglicéridos/metabolismo , Diglicéridos/farmacología , Humanos , Cinética , Lipoproteína Lipasa/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
A series of thioamides were designed as bio-isosteres to the non-nucleoside reverse transcriptase inhibitor trovirdine by replacement of the thiourea NH groups with methylene groups. Eight thioamides were synthesized and in vitro tested for inhibitory effects on the activity of HIV-1 reverse transcriptase wild and mutant types. Three of the 8-thioamides exhibited enzyme inhibitory activities with IC(50) values below 100 microM. While compound (2) exhibited activity against the mutant strain L100I with IC(50) of 70.1 microM, compound (4) showed activity against the mutant strain K103N with IC(50) of 92.7 microM, and compound (8) with activity against the wild type enzyme with IC(50) of 8.9 microM. Each of the three thioamides could serve as a lead compound for further activity optimization.
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Fármacos Anti-VIH/síntesis química , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/síntesis química , Tioamidas/síntesis química , Fármacos Anti-VIH/farmacología , Diseño de Fármacos , Concentración 50 Inhibidora , Mutación , Piridinas , Inhibidores de la Transcriptasa Inversa/farmacología , Relación Estructura-Actividad , Tioamidas/farmacología , Tioamidas/uso terapéuticoRESUMEN
Multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette superfamily. Using the energy provided by ATP hydrolysis, it transports a broad spectrum of substrates across the plasma membrane, including hormones, leukotriene C(4), bile salts, and anti-cancer drugs. Recent works have suggested that P-glycoprotein is associated to cholesterol and sphingolipid-rich membrane microdomains and that cholesterol upregulates its ATPase and drug transport activities. Confocal microscopy experiments and Triton X-100 extraction of detergent-resistant membranes provide evidence that MRP1 is not located in raft-like structures and that its activity is downregulated by cholesterol. The data are discussed in terms of cholesterol-protein interaction and topology.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Octoxinol/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Adenosina Trifosfatasas/metabolismo , Transporte Biológico , Línea Celular , Humanos , Microscopía Confocal , Unión ProteicaRESUMEN
ATP-binding cassette transporters G5 and G8 are half-transporters expressed on the apical membranes of enterocytes and hepatocytes that limit intestinal uptake and promote secretion of neutral sterols. Genetic defects that inactivate either half-transporter cause accumulation of cholesterol and plant sterols, resulting in premature coronary atherosclerosis. These observations suggest that G5 and G8 promote the translocation of sterols across membranes, but the primary transport substrate of the G5G8 complex has not been directly determined. Here we report the development of a sterol transfer assay using "inside-out" membrane vesicles from Sf9 cells expressing recombinant mouse G5 and G8. Radiolabeled cholesterol or sitosterol was transferred from donor liposomes to G5- and G8-containing membrane vesicles in an ATP-dependent and vanadate-sensitive manner; net transfer of cholesterol was associated with an increase in vesicular cholesterol mass. CTP, GTP, and UTP, as well as ATP, supported transfer but with lesser efficiency (ATP >> CTP > GTP > UTP). Transfer was specific for sterols and was stereoselective; minimal ATP-dependent and vanadate-sensitive transfer of cholesteryl oleate, phosphatidylcholine, or enantiomeric cholesterol was observed. These studies indicate that G5 and G8 are sufficient for reconstitution of sterol transfer activity in vitro and provide the first demonstration that sterols are direct transport substrates of the G5 and G8 heterodimer.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Colesterol/metabolismo , Lipoproteínas/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5 , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Transporte Biológico , Dimerización , Lipoproteínas/química , Liposomas/metabolismo , Fusión de Membrana , Ratones , Proteínas Recombinantes/biosíntesis , Spodoptera , Vanadatos/farmacologíaRESUMEN
The structural features of sterols required to support mammalian cell growth have not been fully defined. Here, we use mutant CHO cells that synthesize only small amounts of cholesterol to test the capacity of various sterols to support growth. Sterols with minor modifications of the side chain (e.g., campesterol, beta-sitosterol, and desmosterol) supported long-term growth of mutant cells, but sterols with more complex modifications of the side chain, the sterol nucleus, or the 3-hydroxy group did not. After 60 days in culture, the exogenous sterol comprised >90% of cellular sterols. Inactivation of residual endogenous synthesis with the squalene epoxidase inhibitor NB-598 prevented growth in beta-sitosterol and greatly reduced growth in campesterol. Growth of cells cultured in beta-sitosterol and NB-598 was restored by adding small amounts of cholesterol to the medium. Surprisingly, enantiomeric cholesterol also supported cell growth, even in the presence of NB-598. Thus, sterols fulfill two roles in mammalian cells: (i) a bulk membrane requirement in which phytosterols can substitute for cholesterol and (ii) other processes that specifically require small amounts of cholesterol but are not enantioselective.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Colesterol/metabolismo , Colesterol/farmacología , Animales , Bencilaminas/toxicidad , Células CHO , Colesterol/análogos & derivados , Colesterol/genética , Cricetinae , Cricetulus , Fitosteroles/metabolismo , Fitosteroles/farmacología , Sitoesteroles/metabolismo , Sitoesteroles/farmacología , Tiofenos/toxicidadRESUMEN
The chiral specificity of the interactions of peptides that induce the formation of cholesterol-rich domains has not been extensively investigated. Both the peptide and most lipids are chiral, so there is a possibility that interactions between peptide and lipid could require chiral recognition. On the other hand, in our models with small peptides, the extent of folding of the peptide to form a specific binding pocket is limited. We have determined that replacing cholesterol with its enantiomer, ent-cholesterol, alters the modulation of lipid organization by peptides. The phase-transition properties of SOPC (1-stearoyl-2-oleoylphosphatidylcholine):cholesterol [in a 6:4 ratio with 0.2 mol% PtdIns(4,5)P2] are not significantly altered when ent-cholesterol replaces cholesterol. However, in the presence of 10 mol% of a 19-amino-acid, N-terminally myristoylated fragment (myristoyl-GGKLSKKKKGYNVNDEKAK-amide) of the protein NAP-22 (neuronal axonal membrane protein), the lipid mixture containing cholesterol undergoes separation into cholesterol-rich and cholesterol-depleted domains. This does not occur when ent-cholesterol replaces cholesterol. In another example, when N-acetyl-Leu-Trp-Tyr-Ile-Lys-amide (N-acetyl-LWYIK-amide) is added to SOPC:cholesterol (7:3 ratio), there is a marked increase in the transition enthalpy of the phospholipid, indicating separation of a cholesterol-depleted domain of SOPC. This phenomenon completely disappears when ent-cholesterol replaces cholesterol. The all-D-isomer of N-acetyl-LWYIK-amide also induces the formation of cholesterol-rich domains with natural cholesterol, but does so to a lesser extent with ent-cholesterol. Thus specific peptide chirality is not required for interaction with cholesterol-containing membranes. However, a specific chirality of membrane lipids is required for peptide-induced formation of cholesterol-rich domains.