RESUMO
Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Limoninas/farmacologia , Mitocôndrias/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/genética , Humanos , Limoninas/química , Limoninas/metabolismo , Limoninas/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Chenodeoxycholic acid (CDCA), 3α,7α-dihydroxy-5ß-cholan-24-oic acid, is a primary bile acid generated in the liver from cholesterol. In liver cells CDCA is conjugated with glycine or taurine to form two bile salts, Glyco-CDCA and Tauro-CDCA, before being released into the bile ducts. In the intestine, CDCA is further metabolized to generate a 7ß epimer, i.e., the ursodeoxycholic acid (UDCA), or dehydroxylate to generate lithocolic acid (LCA). In humans, CDCA is the physiological ligand for the bile acid sensor farnesoid X receptor (FXR), while LCA is a potent agonist for a G protein-coupled receptor, known as GPBAR1 (TGR5). Along with UDCA, CDCA has been clinically used for the dissolution of gallbladder stones at doses ranging from 375 to 750 mg/day, with a success rate of 8 to 18%. Because the efficacy of CDCA was significantly lower than that of UDCA and 18-30% of patients developed significant side effects, the most frequent being diarrhea and a reversible increase in aminotransferases plasma levels, this application has lost its therapeutic relevance. Additionally, the combination of CDCA with UDCA, generally at doses of 5-10 mg/kg each, has failed to provide significant advantages over UDCA alone. In 2017, CDCA has been approved as an orphan indication for the treatment of patients with cerebrotendinous xanthomatosis (CTX), a rare autosomal recessive disorder caused by mutations of sterol 27-hydroxylase (CYP27A1) gene. Since CYP27A1 is essential for cholesterol breakdown, CTX patients develop abnormal lipid storage with increased plasma and tissue levels of cholestanol and very low/absent production of CDCA. CDCA is a potent inhibitor of CYP27A1, and early initiation of CDCA therapy, at doses up to 750 mg/day, is considered the standard medical therapy for CTX resulting in decreased plasma levels of cholestanol and stabilization of neurologic symptoms. Studies in CTX patients have also shown that CDCA might suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in the liver. Furthermore, CDCA promotes the release of glucagon-like peptide-1 (GLP-1) in diabetic patients, likely by activating GPBAR1.
Assuntos
Ácido Quenodesoxicólico/uso terapêutico , Xantomatose Cerebrotendinosa , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanol/sangue , Colesterol , Humanos , Fígado , Receptores Citoplasmáticos e Nucleares , Receptores Acoplados a Proteínas GRESUMO
There is an urgent need for more effective strategies to treat ovarian cancer. Elevated cholesterol levels are associated with a decreased progression-free survival time (PFS) while statins are protective. 27-Hydroxycholesterol (27HC), a primary metabolite of cholesterol, has been shown to modulate the activities of the estrogen receptors (ERs) and liver x receptors (LXRs) providing a potential mechanistic link between cholesterol and ovarian cancer progression. We found that high expression of CYP27A1, the enzyme responsible for the synthesis of 27HC, was associated with decreased PFS, while high expression of CYP7B1, responsible for 27HC catabolism, was associated with increased PFS. However, 27HC decreased the cellular proliferation of various ovarian cancer cell lines in an LXR-dependent manner. Intriguingly, ID8 grafts were unable to effectively establish in CYP27A1-/- mice, indicating involvement of the host environment. Tumors from mice treated with 27HC had altered myeloid cell composition, and cells from the marrow stem cell lineage were found to be responsible for the effects in CYP27A1-/- mice. While inhibition of CYP27A1 or immune checkpoint did not significantly alter tumor size, their combination did, thereby highlighting this axis as a therapeutic target.
Assuntos
Colestanotriol 26-Mono-Oxigenase/genética , Neoplasias Ovarianas/genética , Animais , Antígeno B7-H1/antagonistas & inibidores , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/deficiência , Colesterol na Dieta/efeitos adversos , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Hidroxicolesteróis/metabolismo , Camundongos , Células Supressoras Mieloides/citologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , Intervalo Livre de Progressão , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
PURPOSE: 27-hydroxycholesterol (27HC), an endogenous selective estrogen receptor modulator (SERM), drives the growth of estrogen receptor-positive (ER+) breast cancer. 1,25-dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D, is known to inhibit expression of CYP27B1, which is very similar in structure and function to CYP27A1, the synthesizing enzyme of 27HC. Therefore, we hypothesized that 1,25(OH)2D may also inhibit expression of CYP27A1, thereby reducing 27HC concentrations in the blood and tissues that express CYP27A1, including breast cancer tissue. METHODS: 27HC, 25-hydroxyvitamin D (25OHD), and 1,25(OH)2D were measured in sera from 29 breast cancer patients before and after supplementation with low-dose (400 IU/day) or high-dose (10,000 IU/day) vitamin D in the interval between biopsy and surgery. RESULTS: A significant increase (p = 4.3E-5) in 25OHD and a decrease (p = 1.7E-1) in 27HC was observed in high-dose versus low-dose vitamin D subjects. Excluding two statistical outliers, 25OHD and 27HC levels were inversely correlated (p = 7.0E-3). CONCLUSIONS: Vitamin D supplementation can decrease circulating 27HC of breast cancer patients, likely by CYP27A1 inhibition. This suggests a new and additional modality by which vitamin D can inhibit ER+ breast cancer growth, though a larger study is needed for verification.
Assuntos
Neoplasias da Mama/dietoterapia , Colestanotriol 26-Mono-Oxigenase/genética , Hidroxicolesteróis/sangue , Vitamina D/administração & dosagem , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Biópsia , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Linhagem Celular Tumoral , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Suplementos Nutricionais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores de Estrogênio/genética , Moduladores Seletivos de Receptor Estrogênico/administração & dosagemRESUMO
Sterol 27-hydroxylase (CYP27A1) plays an important role in cholesterol homeostasis by degrading cholesterol to bile acids. Betaine can alleviate high-fat diet-induced hepatic cholesterol accumulation and maternal betaine treatment programs the hepatic expression of CYP27A1 in offspring. Excessive corticosterone (CORT) exposure causes hepatic cholesterol deposition in chickens, yet it remains unknown whether prenatal betaine modulates CORT-induced cholesterol accumulation in chicken liver later in life and whether it involves epigenetic gene regulation of CYP27A1. In this study, fertilized eggs were injected with saline or betaine at 2.5mg/egg before incubation, and the hatchlings were raised under the same condition till 56days of age followed by 7days of subcutaneous CORT injection. Plasma concentrations of total cholesterol (Tch), HDL- and LDL-cholesterol were significantly increased (P<0.05), after CORT challenge, in both control and betaine groups. However, prenatal betaine exposure prevented CORT-induced increase (P<0.05) in hepatic Tch content. Hepatic expression of cholesterol biosynthesis genes and ACAT1 protein that esterifies cholesterol for storage, were activated in both control and betaine groups upon CORT challenge. However, betaine-treated chickens were protected from CORT-induced repression (P<0.05) in LXR and CYP27A1 expression in the liver. CORT-induced down-regulation of LXR and CYP27A1 coincided with significantly increased (P<0.05) CpG methylation on their promoters, which was significantly ameliorated in betaine-treated chickens. These results suggest that in ovo betaine injection alleviates CORT-induced hepatic cholesterol deposition most probably through epigenetic regulation of CYP27A1 and LXR genes in juvenile chickens.
Assuntos
Betaína/administração & dosagem , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Corticosterona/farmacologia , Metilação de DNA/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Regiões Promotoras Genéticas/genética , Envelhecimento , Animais , Animais Recém-Nascidos , Anti-Inflamatórios/farmacologia , Western Blotting , Galinhas/crescimento & desenvolvimento , Galinhas/metabolismo , Colestanotriol 26-Mono-Oxigenase/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , Colesterol/metabolismo , Epigênese Genética/efeitos dos fármacos , Feminino , Fármacos Gastrointestinais/administração & dosagem , Imunoprecipitação , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Gravidez , Efeitos Tardios da Exposição Pré-Natal , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Vitamin D metabolism was studied in primary human dermal fibroblasts with focus on drug-mediated gene regulation related to adverse side effects of antiretroviral drugs used in HIV therapy. The fibroblasts expressed mRNA for cytochrome P450 (CYP) enzymes catalysing bioactivating (CYP2R1, CYP27A1 and CYP27B1) and catabolic reactions (CYP24A1). The cells produced both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 . The results demonstrate that primary dermal fibroblasts have an active vitamin D3 -metabolizing system. High incidence of low bone mineral density is a concern for HIV-infected patients treated with antiretroviral drugs. Osteomalacia and severe vitamin D deficiency have been reported. We investigated whether drug-mediated gene regulation could be a possible mechanism behind these adverse drug effects. Fibroblasts were treated with different drugs used in HIV therapy, and the 1α,25-dihydroxyvitamin D3 levels and relative mRNA levels for crucial enzymes were determined. Efavirenz, stavudine and ritonavir significantly down-regulated the bioactivating CYP2R1 and up-regulated the catabolic CYP24A1. The drugs reduced bioactivating enzyme activities and cellular levels of 1α,25-dihydroxyvitamin D3 . The current results indicate that effects on gene expression may lead to disturbed vitamin D metabolism and decreased cellular levels of active vitamin D3 . The data are consistent with the impaired bone health in patients treated with certain antiretroviral drugs.
Assuntos
Fármacos Anti-HIV/farmacologia , Colecalciferol/metabolismo , Colestanotriol 26-Mono-Oxigenase/metabolismo , Família 2 do Citocromo P450/metabolismo , Derme/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Vitamina D3 24-Hidroxilase/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Adolescente , Adulto , Alcinos , Benzoxazinas/farmacologia , Calcifediol/metabolismo , Calcitriol/antagonistas & inibidores , Calcitriol/metabolismo , Células Cultivadas , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/genética , Ciclopropanos , Família 2 do Citocromo P450/antagonistas & inibidores , Família 2 do Citocromo P450/genética , Derme/citologia , Derme/metabolismo , Feminino , Humanos , Masculino , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Ritonavir/farmacologia , Estavudina/farmacologia , Vitamina D3 24-Hidroxilase/química , Vitamina D3 24-Hidroxilase/genética , Adulto JovemRESUMO
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
Local production and action of cholesterol metabolites such as steroids or oxysterols within endocrine tissues are currently recognized as an important principle in the cell type- and tissue-specific regulation of hormone effects. In adipocytes, one of the most abundant endocrine cells in the human body, the de novo production of steroids or oxysterols from cholesterol has not been examined. Here, we demonstrate that essential components of cholesterol transport and metabolism machinery in the initial steps of steroid and/or oxysterol biosynthesis pathways are present and active in adipocytes. The ability of adipocyte CYP11A1 in producing pregnenolone is demonstrated for the first time, rendering adipocyte a steroidogenic cell. The oxysterol 27-hydroxycholesterol (27HC), synthesized by the mitochondrial enzyme CYP27A1, was identified as one of the major de novo adipocyte products from cholesterol and its precursor mevalonate. Inhibition of CYP27A1 activity or knockdown and deletion of the Cyp27a1 gene induced adipocyte differentiation, suggesting a paracrine or autocrine biological significance for the adipocyte-derived 27HC. These findings suggest that the presence of the 27HC biosynthesis pathway in adipocytes may represent a defense mechanism to prevent the formation of new fat cells upon overfeeding with dietary cholesterol.
Assuntos
Adipócitos/metabolismo , Colestanotriol 26-Mono-Oxigenase/metabolismo , Hidroxicolesteróis/metabolismo , Esteroides/biossíntese , Células 3T3-L1 , Adipócitos/citologia , Tecido Adiposo/citologia , Tecido Adiposo/metabolismo , Animais , Vias Biossintéticas/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/genética , Células Hep G2 , Humanos , Immunoblotting , Masculino , Ácido Mevalônico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Pregnenolona/metabolismo , Interferência de RNA , Ratos , Ratos Sprague-DawleyRESUMO
Hypercholesterolemia is a risk factor for estrogen receptor (ER)-positive breast cancers and is associated with a decreased response of tumors to endocrine therapies. Here, we show that 27-hydroxycholesterol (27HC), a primary metabolite of cholesterol and an ER and liver X receptor (LXR) ligand, increases ER-dependent growth and LXR-dependent metastasis in mouse models of breast cancer. The effects of cholesterol on tumor pathology required its conversion to 27HC by the cytochrome P450 oxidase CYP27A1 and were attenuated by treatment with CYP27A1 inhibitors. In human breast cancer specimens, CYP27A1 expression levels correlated with tumor grade. In high-grade tumors, both tumor cells and tumor-associated macrophages exhibited high expression levels of the enzyme. Thus, lowering circulating cholesterol levels or interfering with its conversion to 27HC may be a useful strategy to prevent and/or treat breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Hidroxicolesteróis/metabolismo , Hipercolesterolemia/metabolismo , Animais , Neoplasias da Mama/sangue , Linhagem Celular Tumoral , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Hidroxicolesteróis/antagonistas & inibidores , Hidroxicolesteróis/sangue , Hipercolesterolemia/sangue , Neoplasias Pulmonares/secundário , Camundongos , Células Tumorais CultivadasRESUMO
Glucocorticoid therapy is accompanied by development of processes typical of steroid osteoporosis. Indirect effects of glucocorticoids on the bone tissue are due to changes in mineral metabolism, which is regulated by vitamin D3. In this connection, we studied the influence of prednisolone on cholecalciferol metabolism. The study has shown that prednisolone action causes impairment of cholecalciferol metabolism in hepatocytes due to inhibiting vitamin D3 25-hydroxylase activity. Microsomal (CYP2R1) and mitochondrial (CYP27A1) isoenzymes of vitamin D3 25-hydroxylase were found to function at different concentrations of the substrate. The relative protein contents of the isoenzymes greatly differed in the liver with the prevalence of CYP27A1 over CYP2R1. Prednisolone administration resulted in the lowering of both mitochondrial and microsomal isoenzymes of vitamin D3 25-hydroxylase. The inhibition of vitamin D3 25-hydroxylating system in hepatocytes contributed to a significant reduction in blood serum 25OHD3.
Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Glucocorticoides/farmacologia , Hepatócitos/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Prednisolona/farmacologia , Animais , Hidrocarboneto de Aril Hidroxilases/metabolismo , Calcifediol/sangue , Colecalciferol/metabolismo , Colestanotriol 26-Mono-Oxigenase/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/enzimologia , Hidroxilação , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/enzimologia , RatosRESUMO
A series of (E)-2-(2-substituted benzylidene)- and 2-(2-substituted benzyl)-6-methoxy-tetralones were prepared, using an efficient synthetic scheme, and evaluated for their inhibitory activity against cytochrome P450C24A1 (CYP24A1) hydroxylase. In general the reduced benzyl tetralones were more active than the parent benzylidene tetralones. The 2-ethyl and 2-trifluoromethyl benzyl tetralone derivatives (4c and 4b) showed optimal activity in this series with IC(50) values of 1.92 microM and 2.08 microM, respectively compared with the standard ketoconazole IC(50) 0.52 microM. The 2-bromobenzyl tetralone (4d) showed a preference for CYP27A1 (IC(50) 59 nM) over CYP24A1 (IC50 16.3 microM) and may be a useful lead in CYP27A1 inhibition studies. The 2-ethylphenyl benzyl derivative (9c), which showed weak activity against the wild type CYP24A1 (IC(50) 25.57 microM), exhibited enhanced inhibitory activity towards L148F and M416T mutants, this difference in activity for the L148F mutant has been explained using molecular modelling.
Assuntos
Compostos de Benzilideno/química , Compostos de Benzilideno/farmacologia , Esteroide Hidroxilases/antagonistas & inibidores , Esteroide Hidroxilases/metabolismo , Tetralonas/química , Tetralonas/farmacologia , Animais , Compostos de Benzilideno/síntese química , Linhagem Celular , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , Cricetinae , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Mutação , Esteroide Hidroxilases/genética , Relação Estrutura-Atividade , Tetralonas/síntese química , Vitamina D3 24-HidroxilaseRESUMO
AIMS: In this study, we investigated the impact of enhancing cholesterol delivery to mitochondrial sterol 27-hydroxylase, via steroidogenic acute regulatory protein (StAR), on the expression of genes involved in macrophage cholesterol homeostasis and efflux of cholesterol to apolipoprotein (apo) AI. METHODS AND RESULTS: Stably transfected, murine (RAW 264.7) macrophages were used to investigate the role of StAR in cholesterol homeostasis. Cellular responses were analysed using quantitative PCR, immunoblotting, and an LXRE reporter plasmid; [3H]cholesterol efflux was measured in the presence or absence of apoAI. Macrophage overexpression of mitochondrial cholesterol trafficking protein, StAR, activates and induces expression of liver X receptors (LXRs), and significantly alters expression of genes involved in cholesterol homeostasis, decreasing Fdps, Hmgcr, Mvk, Ldlr, and Scap, and markedly increasing Abca1 mRNA and protein. Overexpression of StAR, but not mutated 'loss-of-function' (R181L) StAR, enhanced efflux of [3H]cholesterol to apoAI, and this effect was maintained in macrophages pretreated with LDL or acetylated LDL. The effect of StAR overexpression on apoAI-dependent [3H]cholesterol efflux was mimicked by non-sterol agonist, T901317, and 27-hydroxycholesterol, and blocked by LXR inhibitor, geranylgeranyl pyrophosphate, sterol 27-hydroxylase inhibitor, GW273297x, and probucol, inhibitor of ATP binding cassette transporter A1 (ABCA1). Importantly, all observed effects of StAR overexpression were dependent upon cyclic AMP (cAMP analogue, dibutyryl cAMP), which is required for the full activity of the StAR protein to be manifested. CONCLUSION: Macrophage overexpression of StAR significantly enhances LXR-dependent apoAI- and ABCA1-dependent cholesterol efflux, by which disposal of excess arterial cholesterol deposits and atheroma regression can be achieved.
Assuntos
Apolipoproteína A-I/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Fosfoproteínas/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Colestanotriol 26-Mono-Oxigenase/metabolismo , CMP Cíclico/análogos & derivados , CMP Cíclico/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Genes Reporter , Homeostase , Hidrocarbonetos Fluorados/farmacologia , Hidroxicolesteróis/metabolismo , Immunoblotting , Metabolismo dos Lipídeos/genética , Lipoproteínas LDL/metabolismo , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Camundongos , Mutação , Receptores Nucleares Órfãos/agonistas , Receptores Nucleares Órfãos/metabolismo , Fosfoproteínas/genética , Fosfatos de Poli-Isoprenil/farmacologia , Probucol/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Transfecção , Triglicerídeos/metabolismo , Regulação para CimaRESUMO
Hyperlipidaemia, i.e. increase in total cholesterol and triglycerides, is a common side-effect of the immunosuppressive drugs rapamycin (RAPA) and cyclosporine A (CsA), and is probably related to inhibition of the 27-hydroxylation of cholesterol (acid pathway of bile acid biosynthesis). This might be one of the causes for the increase in plasma cholesterol, as 27-hydroxycholesterol is a potent suppressor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), a key enzyme of cholesterol synthesis. As the sterol 27-hydroxylase (CYP27A1) inhibition by CsA is well known, we evaluated the effect of another immunosuppressive drug, RAPA, on this enzyme in HepG2 mitochondria, which confirmed the dose-dependent inhibition of mitochondrial CYP27A1 by cyclosporine (10-20 microM), while the inhibition by RAPA required a higher dose (50-100 microM). Corresponding K(i) was 10 microM for CsA (non-competitive inhibition) and 110 microM for RAPA (competitive inhibition). Cotreatment with both immunosuppressive drugs showed an additive inhibitory effect on CYP27A1 activity. Later, we analysed the effect of these immunosuppressants on HMGR expression in HepG2 cells, and a dose-dependent up-regulation of HMGR gene expression was observed. The results suggest that RAPA and CsA are both inhibitors of CYP27A1 activity with slightly different mechanisms and that they may accordingly increase HMGR expression.
Assuntos
Colestanotriol 26-Mono-Oxigenase/metabolismo , Colesterol/biossíntese , Ciclosporina/farmacologia , Hidroximetilglutaril-CoA Redutases/biossíntese , Imunossupressores/farmacologia , Sirolimo/farmacologia , Linhagem Celular Tumoral , Colestanotriol 26-Mono-Oxigenase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Mitocôndrias/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Prolonged therapy with phenobarbital may cause vitamin D deficiency or osteomalacia. In the current study, we propose a novel mechanism for drug-induced osteomalacia involving impaired bioactivation of vitamin D(3) due to decreased 25-hydroxylation of vitamin D(3) in liver. The present data, using the pig as model, demonstrate direct effects by phenobarbital on the expression of CYP27A1 and CYP2D25, two important 25-hydroxylases. Treatment by phenobarbital markedly reduced the rate of 25-hydroxylation by primary hepatocytes and suppressed the cellular CYP27A1 mRNA levels. The rate of 25-hydroxylation by two different purified 25-hydroxylases, microsomal CYP2D25, and mitochondrial CYP27A1, respectively, was dose-dependently inhibited by phenobarbital. Reporter assay experiments in liver-derived HepG2 cells revealed a marked PXR-mediated transcriptional downregulation of the CYP2D25 promoter. In addition, the data indicate that phenobarbital might affect the mRNA stability of CYP2D25. Taken together, the data suggest that vitamin D(3) 25-hydroxylation may be suppressed by phenobarbital. A downregulation of 25-hydroxylation by phenobarbital may explain, at least in part, the increased risk of osteomalacia, bone loss, and fractures in long-term phenobarbital therapy.