RESUMO
Apolipoprotein D (APOD) is an atypical apolipoprotein with unknown significance for retinal structure and function. Conversely, apolipoprotein E (APOE) is a typical apolipoprotein with established roles in retinal cholesterol transport. Herein, we immunolocalized APOD to the photoreceptor inner segments and conducted ophthalmic characterizations of ApoD-/- and ApoD-/-ApoE-/- mice. ApoD-/- mice had normal levels of retinal sterols but changes in the chorioretinal blood vessels and impaired retinal function. The whole-body glucose disposal was impaired in this genotype but the retinal glucose metabolism was unchanged. ApoD-/-ApoE-/- mice had altered sterol profile in the retina but apparently normal chorioretinal vasculature and function. The whole-body glucose disposal and retinal glucose utilization were enhanced in this genotype. OB-Rb, both leptin and APOD receptor, was found to be expressed in the photoreceptor inner segments and was at increased abundance in the ApoD-/- and ApoD-/-ApoE-/- retinas. Retinal levels of Glut4 and Cd36, the glucose transporter and scavenger receptor, respectively, were increased as well, thus linking APOD to retinal glucose and fatty acid metabolism and suggesting the APOD-OB-Rb-GLUT4/CD36 axis. In vivo isotopic labeling, transmission electron microscopy, and retinal proteomics provided additional insights into the mechanism underlying the retinal phenotypes of ApoD-/- and ApoD-/-ApoE-/- mice. Collectively, our data suggest that the APOD roles in the retina are context specific and could determine retinal glucose fluxes into different pathways. APOD and APOE do not play redundant, complementary or opposing roles in the retina, rather their interplay is more complex and reflects retinal responses elicited by lack of these apolipoproteins.
Assuntos
Apolipoproteínas D/metabolismo , Retina/metabolismo , Animais , Apolipoproteínas D/deficiência , Apolipoproteínas D/genética , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Antígenos CD36/metabolismo , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Feminino , Genótipo , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Marcação por Isótopo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteômica , Retina/patologia , Esteróis/análise , Esteróis/metabolismoRESUMO
Cytochrome P450 27A1 (CYP27A1) is a ubiquitous enzyme that hydroxylates cholesterol and other sterols. Complete CYP27A1 deficiency owing to genetic mutations is detrimental to human health, whereas 50% of activity retention is not and does not affect the whole body cholesterol levels. CYP27A1 is considered a potential therapeutic target in breast cancer and age-related neurodegenerative diseases; however, CYP27A1 inhibition should be ≤50%. Herein, 131 pharmaceuticals were tested for their effect on CYP27A1-mediated cholesterol 27-hydroxylation by in vitro enzyme assay. Of them, 14 drugs inhibited CYP27A1 by ≥75% and were evaluated for in vitro binding to the enzyme active site and for inhibition constants. All drugs except one (dasatinib) elicited a spectral response in CYP27A1 and had Ki values for cholesterol 27-hydroxylation either in the submicromolar (clevidipine, delavirdine, etravirine, felodipine, nicardipine, nilotinib, and sorafenib) or low micromolar range (abiratone, candesartan, celecoxib, dasatinib, nilvadipine, nimodipine, and regorafenib). Clevidipine, felodipine, nicardipine, nilvadipine, and nimodipine have the same 1,4-dihydropyridine scaffold and are indicated for hypertension. We used two of these antihypertensives (felodipine and nilvadipine) for administration to mice at a 1-mg/kg of body weight dose, daily, for 7 days. Mouse 27-hydroxycholesterol levels in the plasma, brain, and liver were reduced, whereas tissue levels of total cholesterol were unchanged. Structure-activity relationships within the 1,4-dihydropyridine scaffold were investigated, and features important for CY27A1 inhibition were identified. We confirmed our previous finding that CYP27A1 is a druggable enzyme and found additional drugs as well as the scaffold with potential for partial CYP27A1 inhibition in humans.
Assuntos
Anti-Hipertensivos/farmacologia , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Inibidores das Enzimas do Citocromo P-450/farmacologia , Hidroxicolesteróis/metabolismo , Animais , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Felodipino/análogos & derivados , Felodipino/farmacologia , Feminino , Camundongos Endogâmicos C57BLRESUMO
Cytochrome P450 CYP27A1 is the only enzyme in humans converting cholesterol to 27-hydroxycholesterol, an oxysterol of multiple functions, including tissue-specific modulation of estrogen and liver X receptors. Both receptors seem to mediate adverse effects of 27-hydroxycholesterol in breast cancer when the levels of this oxysterol are elevated. The present work assessed druggability of CYP27A1 as a potential antibreast cancer target. We selected 26 anticancer and noncancer medications, most approved by the Food and Drug Administration, and evaluated them first in vitro for inhibition of purified recombinant CYP27A1 and binding to the enzyme active site. Six strong CYP27A1 inhibitors/binders were identified. These were the two antibreast cancer pharmaceuticals anastrozole and fadrozole, antiprostate cancer drug bicalutamide, sedative dexmedetomidine, and two antifungals ravuconazole and posaconazole. Anastrozole was then tested in vivo on mice, which received subcutaneous drug injections for 1 week. Mouse plasma and hepatic 27-hydroxycholesterol levels were decreased 2.6- and 1.6-fold, respectively, whereas plasma and hepatic cholesterol content remained unchanged. Thus, pharmacologic CYP27A1 inhibition is possible in the whole body and individual organs, but does not negatively affect cholesterol elimination. Our results enhance the potential of CYP27A1 as an antibreast cancer target, could be of importance for the interpretation of Femara versus Anastrozole Clinical Evaluation Trial, and bring attention to posaconazole as a potential complementary anti-breast cancer medication. More medications on the US market may have unanticipated off-target inhibition of CYP27A1, and we propose strategies for their identification.
Assuntos
Adjuvantes Farmacêuticos/farmacologia , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Hipnóticos e Sedativos/farmacologia , Adjuvantes Farmacêuticos/química , Anastrozol , Anilidas/química , Anilidas/farmacologia , Animais , Antifúngicos/química , Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Dexmedetomidina/química , Dexmedetomidina/farmacologia , Inibidores Enzimáticos/química , Fadrozol/química , Fadrozol/farmacologia , Feminino , Hipnóticos e Sedativos/química , Camundongos , Camundongos Endogâmicos C57BL , Nitrilas/química , Nitrilas/farmacologia , Ligação Proteica , Tiazóis/química , Tiazóis/farmacologia , Compostos de Tosil/química , Compostos de Tosil/farmacologia , Triazóis/química , Triazóis/farmacologiaRESUMO
Of the different oxygenated cholesterol metabolites, 7-ketocholesterol (7KCh) is considered a noxious oxy-sterol implicated in the development of certain pathologies, including those found in the eye. Here we elucidated whether sterol 27-hydroxylase cytochrome P450 27A1 (CYP27A1) is involved in elimination of 7KCh from the posterior part of the eye: the neural retina and underlying retinal pigment epithelium (RPE). We first established that the affinities of purified recombinant CYP27A1 for 7KCh and its endogenous substrate cholesterol are similar, yet 7KCh is metabolized at a 4-fold higher rate than cholesterol in the reconstituted system in vitro. Lipid extracts from bovine neural retina and RPE were then analyzed by isotope dilution GC-MS for the presence of the 7KCh-derived oxysterols. Two metabolites, 3ß,27-dihydroxy-5-cholesten-7-one (7KCh-27OH) and 3ß-hydroxy-5-cholesten-7-one-26-oic acid (7KCh-27COOH), were detected in the RPE but not in the neural retina. 7KCh-27OH was also formed when RPE homogenates were supplemented with NADPH and the mitochondrial redox system. Quantifications in human RPE showed that CYP27A1 is indeed expressed in the RPE at 2-4-fold higher levels than in the neural retina. The data obtained represent evidence for the role of CYP27A1 in retinal metabolism of 7KCh and suggest that, in addition to cholesterol removal, the functions of this enzyme could also include elimination of toxic endogenous compounds.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Células Epiteliais/metabolismo , Cetocolesteróis , Neurônios/metabolismo , Proteínas Recombinantes/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Ácidos/metabolismo , Animais , Bovinos , Extratos Celulares/química , Colestanotriol 26-Mono-Oxigenase/genética , Sistema Enzimático do Citocromo P-450/genética , Células Epiteliais/citologia , Oftalmopatias/metabolismo , Oftalmopatias/patologia , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Cetocolesteróis/metabolismo , Mitocôndrias/química , Mitocôndrias/metabolismo , Neurônios/citologia , Oxirredução , Proteínas Recombinantes/genética , Epitélio Pigmentado da Retina/citologia , Especificidade por SubstratoRESUMO
Human cytochrome P450 46A1 (CYP46A1) is one of the key enzymes in cholesterol homeostasis in the brain. The crystallization and heavy-atom structure solution of an active truncated CYP46A1 in complex with the high-affinity substrate analogue cholesterol-3-sulfate (CH-3S) is reported. The 2.6 angstroms structure of CYP46A1-CH-3S was solved using both anion and cation heavy-atom salts. In addition to the native anomalous signal from the haem iron, an NaI anion halide salt derivative and a complementary CsCl alkali-metal cation salt derivative were used. The general implications of the use of halide and alkali-metal quick soaks are discussed. The importance of using isoionic strength buffers, the titration of heavy-atom salts into different ionic species and the role of concentration are considered. It was observed that cation/anion-binding sites will occasionally overlap, which could negatively impact upon mixed RbBr soaks used for multiple anomalous scatterer MAD (MMAD). The use of complementary cation and anion heavy-atom salt derivatives is a convenient and powerful tool for MIR(AS) structure solution.