RESUMO
Regioselective transformations at similar functional groups are of paramount importance in organic synthesis. Traditional strategies towards regioselective functionalization include serial protection/deprotection and sequential synthesis. Modern organic synthesis emphasizes pathway efficiency and protecting group free routes with a goal of exploiting inherent differences in reactivity. This study reports a method for the regioselective functionalization of anilines over aliphatic amines. Utilizing classic conditions for the Baeyer-Mills reaction, anilines were shown to react preferentially in the presence of aliphatic amines. Subsequently, this principle of reactivity was extended to other electrophiles and conditions.
RESUMO
The γ-amino-α-aryl alcohol is a key functional group for the design of inhibitors directed toward a critical family of metabolic enzymes. Here we report the transformation of simple aryl halides to a highly functionalized benzyl (3-oxo-3-arylpropyl)carbamate intermediate that can rapidly be converted to a high value γ-amino-α-aryl alcohol. This chemistry is realized through a two-step process involving an enamine-based Heck coupling (EBHC) followed by a one-pot catalytic Cbz-deprotection and ketone reduction of EBHC products.
Assuntos
Amino Álcoois , Cetonas , Catálise , Estrutura MolecularRESUMO
Vertebrate rhodopsin (Rh) contains 11-cis-retinal as a chromophore to convert light energy into visual signals. On absorption of light, 11-cis-retinal is isomerized to all-trans-retinal, constituting a one-way reaction that activates transducin (Gt) followed by chromophore release. Here we report that bovine Rh, regenerated instead with a six-carbon-ring retinal chromophore featuring a C11=C12 double bond locked in its cis conformation (Rh6mr), employs an atypical isomerization mechanism by converting 11-cis to an 11,13-dicis configuration for prolonged Gt activation. Time-dependent UV-vis spectroscopy, HPLC, and molecular mechanics analyses revealed an atypical thermal reisomerization of the 11,13-dicis to the 11-cis configuration on a slow timescale, which enables Rh6mr to function in a photocyclic manner similar to that of microbial Rhs. With this photocyclic behavior, Rh6mr repeatedly recruits and activates Gt in response to light stimuli, making it an excellent candidate for optogenetic tools based on retinal analog-bound vertebrate Rhs. Overall, these comprehensive structure-function studies unveil a unique photocyclic mechanism of Rh activation by an 11-cis-to-11,13-dicis isomerization.
Assuntos
Rodopsina/química , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Isomerismo , Processos Fotoquímicos , Rodopsina/fisiologia , Rodopsina/efeitos da radiaçãoRESUMO
Catalytic Z-isomerization of retinoids to their thermodynamically less stable Z-isomer remains a challenge. In this report, we present a photochemical approach for the catalytic Z-isomerization of retinoids using monochromatic wavelength UV irradiation treatment. We have developed a straightforward approach for the synthesis of Z-retinoids in high yield, overcoming common obstacles normally associated with their synthesis. Calculations based on density functional theory (DFT) have allowed us to correlate the experimentally observed Z-isomer distribution of retinoids with the energies of chemically important intermediates, which include ground- and excited-state potential energy surfaces. We also demonstrate the application of the current method by synthesizing gram-scale quantities of 9-cis-retinyl acetate 9Z-a. Operational simplicity and gram-scale ability make this chemistry a very practical solution to the problem of Z-isomer retinoid synthesis.
Assuntos
Complexos de Coordenação/química , Irídio/química , Retinoides/química , Catálise , Teoria da Densidade Funcional , Estrutura Molecular , Processos Fotoquímicos , Estereoisomerismo , Raios UltravioletaRESUMO
Celastrol is an anti-inflammatory natural triterpenoid, isolated from the herb Tripterygium wilfordii or thunder god vine. Here, we define mechanisms mediating anti-inflammatory activity of celastrol and demonstrate efficacy of a dietary celastrol supplement for chemoprevention of inflammation-driven carcinogenesis in mice. Dietary celastrol (31.25 ppm in rodent diet from 8 weeks to 25 weeks of age) is well tolerated and protects against LPS-induced acute inflammation in C57BL/6 mice, potently suppressing LPS-induction of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, Interleukin (IL)-6 and IL-1ß. To test whether dietary celastrol suppresses inflammation-driven colorectal cancer (CRC), we employed a unique model of spontaneous, inflammation-driven CRC in mice harboring a germ line deletion of the p27Kip1 gene and a T cell-specific deletion of Smad4 gene (Smad4co/co;Lck-crep27Kip1-/-or DKO), which develop severe intestinal inflammation and carcinogenesis as early as 3 months of age. Exposure of DKO mice to daily dietary celastrol (12.5 ppm in diet) from 6 weeks of age significantly suppressed development of colitis-associated CRC (CAC). Celastrol chemoprevention of CAC in this new model of intestinal neoplasia was associated with significant suppression of iNOS at 4 months of age, and iNOS, COX-2 and NFκB at 6 months of age, with significant reduction in inflammatory cytokines, IL-6 and IL-1ß. Chemoprevetion of CAC by dietary celastrol was further confirmed in the model of azoxymethane (AOM) plus dextran sodium sulfate (DSS)-induced carcinogenesis in C57BL/6 mice. These data suggest the potential for celastrol as a safe and effective dietary supplement in the chemoprevention of CAC in humans.
Assuntos
Anti-Inflamatórios/farmacologia , Carcinogênese/efeitos dos fármacos , Neoplasias Colorretais/patologia , Suplementos Nutricionais , Triterpenos/farmacologia , Animais , Carcinógenos/toxicidade , Colite/complicações , Neoplasias Colorretais/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Triterpenos PentacíclicosRESUMO
Carotenoid cleavage oxygenases (CCOs) are non-heme iron enzymes that catalyze scission of alkene groups in carotenoids and stilbenoids to form biologically important products. CCOs possess a rare four-His iron center whose resting-state structure and interaction with substrates are incompletely understood. Here, we address this knowledge gap through a comprehensive structural and spectroscopic study of three phyletically diverse CCOs. The crystal structure of a fungal stilbenoid-cleaving CCO, CAO1, reveals strong similarity between its iron center and those of carotenoid-cleaving CCOs, but with a markedly different substrate-binding cleft. These enzymes all possess a five-coordinate high-spin Fe(II) center with resting-state Fe-His bond lengths of â¼2.15 Å. This ligand set generates an iron environment more electropositive than those of other non-heme iron dioxygenases as observed by Mössbauer isomer shifts. Dioxygen (O2) does not coordinate iron in the absence of substrate. Substrates bind away (â¼4.7 Å) from the iron and have little impact on its electronic structure, thus excluding coordination-triggered O2 binding. However, substrate binding does perturb the spectral properties of CCO Fe-NO derivatives, indicating proximate organic substrate and O2-binding sites, which might influence Fe-O2 interactions. Together, these data provide a robust description of the CCO iron center and its interactions with substrates and substrate mimetics that illuminates commonalities as well as subtle and profound structural differences within the CCO family.
Assuntos
Alcenos/química , Dioxigenases/química , Heme/química , Conformação ProteicaRESUMO
Propionate, 3-hydroxypropionate (3HP), methylcitrate, related compounds, and ammonium accumulate in body fluids of patients with disorders of propionyl-CoA metabolism, such as propionic acidemia. Although liver transplantation alleviates hyperammonemia, high concentrations of propionate, 3HP, and methylcitrate persist in body fluids. We hypothesized that conserved metabolic perturbations occurring in transplanted patients result from the simultaneous presence of propionate and 3HP in body fluids. We investigated the inter-relations of propionate and 3HP metabolism in perfused livers from normal rats using metabolomic and stable isotopic technologies. In the presence of propionate, 3HP, or both, we observed the following metabolic perturbations. First, the citric acid cycle (CAC) is overloaded but does not provide sufficient reducing equivalents to the respiratory chain to maintain the homeostasis of adenine nucleotides. Second, there is major CoA trapping in the propionyl-CoA pathway and a tripling of liver total CoA within 1 h. Third, liver proteolysis is stimulated. Fourth, propionate inhibits the conversion of 3HP to acetyl-CoA and its oxidation in the CAC. Fifth, some propionate and some 3HP are converted to nephrotoxic maleate by different processes. Our data have implications for the clinical management of propionic acidemia. They also emphasize the perturbations of the liver intermediary metabolism induced by supraphysiological, i.e., millimolar, concentrations of labeled propionate used to trace the intermediary metabolism, in particular, inhibition of CAC flux and major decreases in the [ATP]/[ADP] and [ATP]/[AMP] ratios.
Assuntos
Acil Coenzima A/metabolismo , Ácido Láctico/análogos & derivados , Fígado/metabolismo , Propionatos/metabolismo , Compostos de Amônio/metabolismo , Animais , Isótopos de Carbono , Citratos/metabolismo , Ciclo do Ácido Cítrico , Ácido Láctico/metabolismo , Transplante de Fígado , Masculino , Oxirredução , Acidemia Propiônica/metabolismo , Acidemia Propiônica/cirurgia , Proteólise , Ratos , Ratos Sprague-DawleyRESUMO
Modulators of the visual cycle have been developed for treatment of various retinal disorders. These agents were designed to inhibit retinoid isomerase [retinal pigment epithelium-specific 65 kDa protein (RPE65)], the rate-limiting enzyme of the visual cycle, based on the idea that attenuation of visual pigment regeneration could reduce formation of toxic retinal conjugates. Of these agents, certain ones that contain primary amine groups can also reversibly form retinaldehyde Schiff base adducts, which contributes to their retinal protective activity. Direct inhibition of RPE65 as a therapeutic strategy is complicated by adverse effects resulting from slowed chromophore regeneration, whereas effective retinal sequestration can require high drug doses with potential off-target effects. We hypothesized that the RPE65-emixustat crystal structure could help guide the design of retinaldehyde-sequestering agents with varying degrees of RPE65 inhibitory activity. We found that addition of an isopropyl group to the central phenyl ring of emixustat and related compounds resulted in agents effectively lacking in vitro retinoid isomerase inhibitory activity, whereas substitution of the terminal 6-membered ring with branched moieties capable of stronger RPE65 interaction potentiated inhibition. The isopropyl derivative series produced discernible visual cycle suppression in vivo, albeit much less potently than compounds with a high affinity for the RPE65 active site. These agents were distributed into the retina and formed Schiff base adducts with retinaldehyde. Except for one compound [3-amino-1-(3-isopropyl-5-((2,6,6-trimethylcyclohex-1-en-1-yl)methoxy)phenyl)propan-1-ol (MB-007)], these agents conferred protection against retinal phototoxicity, suggesting that both direct RPE65 inhibition and retinal sequestration are mechanisms of potential therapeutic relevance.
Assuntos
Visão Ocular/efeitos dos fármacos , cis-trans-Isomerases/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Sítios de Ligação , Bovinos , Dermatite Fototóxica/prevenção & controle , Feminino , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microssomos/enzimologia , Modelos Moleculares , Epitélio Pigmentado Ocular/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Doenças Retinianas/prevenção & controle , Epitélio Pigmentado da Retina/efeitos dos fármacos , Bases de Schiff/química , cis-trans-Isomerases/química , cis-trans-Isomerases/genética , cis-trans-Isomerases/isolamento & purificação , cis-trans-Isomerases/metabolismoRESUMO
Visual function in vertebrates is dependent on the membrane-bound retinoid isomerase RPE65, an essential component of the retinoid cycle pathway that regenerates 11-cis-retinal for rod and cone opsins. The mechanism by which RPE65 catalyzes stereoselective retinoid isomerization has remained elusive because of uncertainty about how retinoids bind to its active site. Here we present crystal structures of RPE65 in complex with retinoid-mimetic compounds, one of which is in clinical trials for the treatment of age-related macular degeneration. The structures reveal the active site retinoid-binding cavity located near the membrane-interacting surface of the enzyme as well as an Fe-bound palmitate ligand positioned in an adjacent pocket. With the geometry of the RPE65-substrate complex clarified, we delineate a mechanism of catalysis that reconciles the extensive biochemical and structural research on this enzyme. These data provide molecular foundations for understanding a key process in vision and pharmacological inhibition of RPE65 with small molecules.
Assuntos
Epitélio Pigmentado da Retina/enzimologia , Retinoides/farmacologia , Visão Ocular/efeitos dos fármacos , cis-trans-Isomerases/antagonistas & inibidores , cis-trans-Isomerases/química , Animais , Sítios de Ligação , Biocatálise , Cristalografia por Raios X , Diterpenos/síntese química , Diterpenos/química , Diterpenos/farmacologia , Ligantes , Luz , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Estrutura Molecular , Palmitatos , Éteres Fenílicos/síntese química , Éteres Fenílicos/química , Éteres Fenílicos/farmacologia , Propanolaminas/síntese química , Propanolaminas/química , Propanolaminas/farmacologia , Ligação Proteica , Conformação Proteica , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos da radiação , Retinoides/síntese química , Retinoides/química , Estereoisomerismo , Visão Ocular/fisiologia , Visão Ocular/efeitos da radiaçãoRESUMO
Oxidative stress triggers the peroxidation of ω-6-polyunsaturated fatty acids to reactive lipid fragments, including (2E)-4-hydroxy-2-nonenal (HNE). We previously reported two parallel catabolic pathways of HNE. In this study, we report a novel metabolite that accumulates in rat liver perfused with HNE or 4-hydroxynonanoic acid (HNA), identified as 3-(5-oxotetrahydro-2-furanyl)propanoyl-CoA. In experiments using a combination of isotopic analysis and metabolomics studies, three catabolic pathways of HNE were delineated following HNE conversion to HNA. (i) HNA is ω-hydroxylated to 4,9-dihydroxynonanoic acid, which is subsequently oxidized to 4-hydroxynonanedioic acid. This is followed by the degradation of 4-hydroxynonanedioic acid via ß-oxidation originating from C-9 of HNA breaking down to 4-hydroxynonanedioyl-CoA, 4-hydroxyheptanedioyl-CoA, or its lactone, 2-hydroxyglutaryl-CoA, and 2-ketoglutaric acid entering the citric acid cycle. (ii) ω-1-hydroxylation of HNA leads to 4,8-dihydroxynonanoic acid (4,8-DHNA), which is subsequently catabolized via two parallel pathways we previously reported. In catabolic pathway A, 4,8-DHNA is catabolized to 4-phospho-8-hydroxynonanoyl-CoA, 3,8-dihydroxynonanoyl-CoA, 6-hydroxyheptanoyl-CoA, 4-hydroxypentanoyl-CoA, propionyl-CoA, and acetyl-CoA. (iii) The catabolic pathway B of 4,8-DHNA leads to 2,6-dihydroxyheptanoyl-CoA, 5-hydroxyhexanoyl-CoA, 3-hydroxybutyryl-CoA, and acetyl-CoA. Both in vivo and in vitro experiments showed that HNE can be catabolically disposed via ω- and ω-1-oxidation in rat liver and kidney, with little activity in brain and heart. Dietary experiments showed that ω- and ω-1-hydroxylation of HNA in rat liver were dramatically up-regulated by a ketogenic diet, which lowered HNE basal level. HET0016 inhibition and mRNA expression level suggested that the cytochrome P450 4A are main enzymes responsible for the NADPH-dependent ω- and ω-1-hydroxylation of HNA/HNE.
Assuntos
Aldeídos/metabolismo , Dieta Cetogênica , Fígado/enzimologia , Oxigênio/metabolismo , Acil Coenzima A/metabolismo , Animais , Encéfalo/metabolismo , Citocromo P-450 CYP4A/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Hidroxilação , Rim/metabolismo , Peroxidação de Lipídeos , Masculino , Espectrometria de Massas , Metabolismo , Miocárdio/metabolismo , Estresse Oxidativo , Perfusão , Ratos , Ratos Sprague-DawleyRESUMO
We recently reported that levulinate (4-ketopentanoate) is converted in the liver to 4-hydroxypentanoate, a drug of abuse, and that the formation of 4-hydroxypentanoate is stimulated by ethanol oxidation. We also identified 3 parallel ß-oxidation pathways by which levulinate and 4-hydroxypentanoate are catabolized to propionyl-CoA and acetyl-CoA. We now report that levulinate forms three seven-carbon cyclical CoA esters by processes starting with the elongation of levulinyl-CoA by acetyl-CoA to 3,6-diketoheptanoyl-CoA. The latter γ-diketo CoA ester undergoes two parallel cyclization processes. One process yields a mixture of tautomers, i.e., cyclopentenyl- and cyclopentadienyl-acyl-CoAs. The second cyclization process yields a methyl-pyrrolyl-acetyl-CoA containing a nitrogen atom derived from the ε-nitrogen of lysine but without carbons from lysine. The cyclic CoA esters were identified in rat livers perfused with levulinate and in livers and brains from rats gavaged with calcium levulinate ± ethanol. Lastly, 3,6-diketoheptanoyl-CoA, like 2,5-diketohexane, pyrrolates free lysine and, presumably, lysine residues from proteins. This may represent a new pathway for protein pyrrolation. The cyclic CoA esters and related pyrrolation processes may play a role in the toxic effects of 4-hydroxypentanoate.
Assuntos
Coenzima A/metabolismo , Inibidores Enzimáticos/metabolismo , Ácidos Levulínicos/metabolismo , Pró-Fármacos/metabolismo , Animais , Encéfalo/metabolismo , Coenzima A/química , Ciclização , Inibidores Enzimáticos/química , Ácidos Levulínicos/química , Fígado/metabolismo , Masculino , Metabolômica , Pró-Fármacos/química , Ratos , Ratos Sprague-DawleyRESUMO
Triterpenoids comprise a very diverse family of polycyclic molecules that is well-known to possess a myriad of medicinal properties. Therefore, triterpenoids constitute an attractive target for medicinal chemistry and diversity-oriented synthesis. Photochemical transformations provide a promising tool for the rapid, green, and inexpensive generation of skeletal diversity in the construction of natural product-like libraries. With this in mind, we have developed a diversity-oriented strategy, whereby the parent triterpenoids bryonolic acid and lanosterol are converted to the pseudosymmetrical polyketones by sequential allylic oxidation and oxidative cleavage of the bridging double bond at the B/C ring fusion. The resultant polyketones were hypothesized to undergo divergent Norrish-Yang cyclization to produce unique 6/4/8-fused triterpenoid analogues. The subtle differences between parent triterpenoids led to dramatically different spatial arrangements of reactive functionalities. This finding was rationalized through conformational analysis to explain unanticipated photoinduced pinacolization, as well as the regio- and stereochemical outcome of the desired Norrish-Yang cyclization.
Assuntos
Triterpenos/química , Ciclização , Conformação Molecular , Fotoquímica , EstereoisomerismoRESUMO
In an effort to access biologically relevant chemical space, a complex natural product derived nonsymmetrical diketone was prepared as a substrate for divergent transannular aldol reactions. The use of common aldol conditions resulted in predominant syn-addition via pathway a, while the use of alumina provided access to the anti-adduct. Screening of a range of Lewis acids of varying strength unexpectedly resulted in the formation of aldol products with 6/7/5/5-fused molecular skeleton via pathway b.
Assuntos
Cetonas/química , Lanosterol/química , Triterpenos/síntese química , Conformação Molecular , Estereoisomerismo , Triterpenos/químicaRESUMO
The remodeling of a natural product core framework by means of diversity-oriented synthesis (DOS) is a valuable approach to access diverse/biologically relevant chemical space and to overcome the limitations of combinatorial-type compounds. Here we provide proof of principle and a thorough conformational analysis for a general strategy whereby the inherent complexity of a starting material is used to define the regio- and stereochemical outcomes of reactions in chemical library construction. This is in contrast to the traditional DOS logic employing reaction development and catalysis to drive library diversity.
Assuntos
Técnicas de Química Combinatória/métodos , Triterpenos/química , Triterpenos/síntese química , Catálise , Técnicas de Química Combinatória/estatística & dados numéricos , Conformação Molecular , Bibliotecas de Moléculas PequenasRESUMO
GHB (γ-hydroxybutyrate) is both a neurotransmitter and a drug of abuse (date-rape drug). We investigated the catabolism of this compound in perfused rat livers. Using a combination of metabolomics and mass isotopomer analysis, we showed that GHB is metabolized by multiple processes, in addition to its previously reported metabolism in the citric acid cycle via oxidation to succinate. A substrate cycle operates between GHB and γ-aminobutyrate via succinic semialdehyde. Also, GHB undergoes (i) ß-oxidation to glycolyl-CoA+acetyl-CoA, (ii) two parallel processes which remove C-1 or C-4 of GHB and form 3-hydroxypropionate from C-2+C-3+C-4 or from C-1+C-2+C-3 of GHB, and (iii) degradation to acetyl-CoA via 4-phosphobutyryl-CoA. The present study illustrates the potential of the combination of metabolomics and mass isotopomer analysis for pathway discovery.
Assuntos
Fígado/metabolismo , Perfusão , Oxibato de Sódio/metabolismo , Animais , Fígado/enzimologia , Perfusão/métodos , Ratos , Ratos Sprague-Dawley , Especificidade por SubstratoRESUMO
In the eye, the isomerization of all-trans-retinal to 11-cis-retinal is accomplished by a metabolic pathway termed the visual cycle that is critical for vision. RPE65 is the essential trans-cis isomerase of this pathway. Emixustat, a retinoid-mimetic RPE65 inhibitor, was developed as a therapeutic visual cycle modulator and used for the treatment of retinopathies. However, pharmacokinetic liabilities limit its further development including: (1) metabolic deamination of the γ-amino-α-aryl alcohol, which mediates targeted RPE65 inhibition, and (2) unwanted long-lasting RPE65 inhibition. We sought to address these issues by more broadly defining the structure-activity relationships of the RPE65 recognition motif via the synthesis of a family of novel derivatives, which were tested in vitro and in vivo for RPE65 inhibition. We identified a potent secondary amine derivative with resistance to deamination and preserved RPE65 inhibitory activity. Our data provide insights into activity-preserving modifications of the emixustat molecule that can be employed to tune its pharmacological properties.
Assuntos
Propanolaminas , Retinoides , Retinoides/farmacologia , Retinoides/metabolismo , Éteres Fenílicos/farmacologia , Visão Ocular , Retinaldeído/metabolismo , Proteínas do OlhoRESUMO
Metabolomics is a data-based research strategy, the aims of which are to identify biomarker pictures of metabolic systems and metabolic perturbations and to formulate hypotheses to be tested. It involves the assay by mass spectrometry or NMR of many metabolites present in the biological system investigated. In this minireview, we outline studies in which metabolomics led to useful biomarkers of metabolic processes. We also illustrate how the discovery potential of metabolomics is enhanced by associating it with stable isotopic techniques.
Assuntos
Biomarcadores/metabolismo , Metaboloma/fisiologia , Metabolômica/métodos , Animais , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Metabolômica/tendênciasRESUMO
Calcium levulinate (4-ketopentanoate) is used as an oral and parenteral source of calcium. We hypothesized that levulinate is converted in the liver to 4-hydroxypentanoate, a new drug of abuse, and that this conversion is accelerated by ethanol oxidation. We confirmed these hypotheses in live rats, perfused rat livers, and liver subcellular preparations. Levulinate is reduced to (R)-4-hydroxypentanoate by a cytosolic and a mitochondrial dehydrogenase, which are NADPH- and NADH-dependent, respectively. A mitochondrial dehydrogenase or racemase system also forms (S)-4-hydroxypentanoate. In livers perfused with [(13)C(5)]levulinate, there was substantial CoA trapping in levulinyl-CoA, 4-hydroxypentanoyl-CoA, and 4-phosphopentanoyl-CoA. This CoA trapping was increased by ethanol, with a 6-fold increase in the concentration of 4-phosphopentanoyl-CoA. Levulinate is catabolized by 3 parallel pathways to propionyl-CoA, acetyl-CoA, and lactate. Most intermediates of the 3 pathways were identified by mass isotopomer analysis and metabolomics. The production of 4-hydroxypentanoate from levulinate and its stimulation by ethanol is a potential public health concern.
Assuntos
Cálcio/farmacologia , Inibidores Enzimáticos/farmacocinética , Ácidos Levulínicos/farmacocinética , Fígado/enzimologia , Ácidos Pentanoicos/metabolismo , Transtornos Relacionados ao Uso de Substâncias , Animais , Depressores do Sistema Nervoso Central/farmacologia , Citoplasma/enzimologia , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacologia , Etanol/farmacologia , Ácidos Levulínicos/efeitos adversos , Ácidos Levulínicos/farmacologia , Masculino , Mitocôndrias Hepáticas/enzimologia , Oxirredução , Ácidos Pentanoicos/efeitos adversos , Perfusão , Ratos , Ratos Sprague-DawleyRESUMO
Bryonolic acid (BA) (1) is a naturally occurring triterpenoid with pleiotropic properties. This study characterizes the mechanisms mediating the anti-inflammatory and antioxidant activities of BA and validates the utility of BA as a tool to explore the relationships between triterpenoid structure and activity. BA reduces the inflammatory mediator NO by suppressing the expression of the inflammatory enzyme inducible nitric oxide synthase (iNOS) in LPS-activated RAW 264.7 macrophage cells. In addition, BA robustly induces the antioxidant protein heme oxygenase-1 (HO-1) in vitro and in vivo in an Nrf2-dependent manner. Further analyses of Nrf2 target genes reveal selectivity for the timing and level of gene induction by BA in treated macrophages with distinct patterns for Nrf2-regulated antioxidant genes. Additionally, the distinct expression profile of BA on Nrf2 target genes relative to oleanolic acid suggests the importance of the triterpenoid scaffold in dictating the pleiotropic effects exerted by these molecules.
Assuntos
Antioxidantes/farmacologia , Heme Oxigenase-1/efeitos dos fármacos , Macrófagos/fisiologia , Triterpenos/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Relação Dose-Resposta a Droga , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 1 Relacionado a NF-E2/efeitos dos fármacos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/análise , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Triterpenos/químicaRESUMO
Rhodopsin-associated (RHO-associated) retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified nonretinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules toward the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Furthermore, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL), indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that nonretinoid chaperones are promising drug candidates in treating RHO-associated RP.