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1.
Eur J Pharm Sci ; 131: 177-194, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30776468

RESUMO

Microsomal cytochrome P450 (CYP) enzymes, isolated from recombinant bacterial/insect/yeast cells, are extensively used for drug metabolism studies. However, they may not always portray how a developmental drug would behave in human cells with intact intracellular transport mechanisms. This study emphasizes the usefulness of human HEK293 kidney cells, grown in 'suspension' for expression of CYPs, in finding potent CYP1A1/CYP1B1 inhibitors, as possible anticancer agents. With live cell-based assays, quinazolinones 9i/9b were found to be selective CYP1A1/CYP1B1 inhibitors with IC50 values of 30/21 nM, and > 150-fold selectivity over CYP2/3 enzymes, whereas they were far less active using commercially-available CYP1A1/CYP1B1 microsomal enzymes (IC50, >10/1.3-1.7 µM). Compound 9i prevented CYP1A1-mediated benzo[a]pyrene-toxicity in normal fibroblasts whereas 9b completely reversed cisplatin resistance in PC-3/prostate, COR-L23/lung, MIAPaCa-2/pancreatic and LS174T/colon cancer cells, underlining the human-cell-assays' potential. Our results indicate that the most potent CYP1A1/CYP1B1 inhibitors would not have been identified if one had relied merely on microsomal enzymes.


Assuntos
Citocromo P-450 CYP1A1 , Citocromo P-450 CYP1B1 , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Quinazolinonas , Antineoplásicos/farmacologia , Benzo(a)pireno/toxicidade , Bioensaio , Linhagem Celular , Cisplatino/farmacologia , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/antagonistas & inibidores , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Quinazolinonas/química , Quinazolinonas/farmacologia
2.
Biochim Biophys Acta Gen Subj ; 1863(2): 291-303, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30278240

RESUMO

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most difficult to biodegradate and the most toxic dioxin congener. Previously, we demonstrated in silico the ability of pig CYP1A1 to hydroxylate 2,7-dichlorodibenzo-p-dioxin (DiCDD), but not TCDD. To increase our knowledge concerning the low effectiveness of TCDD biodegradability, we analyzed in silico the binding selectivity and affinity between pig CYP1B1 and the two dioxins by means of molecular modeling. We also compared the effects of TCDD and DiCDD on CYP1B1 gene expression (qRT-PCR) and catalytic (EROD) activity in porcine granulosa cells. It was found that DiCDD and TCDD were stabilized within the pig CYP1B1 active site by hydrophobic interactions. The analysis of substrate channel availability revealed that both dioxins opened the exit channel S, allowing metabolites to leave the enzyme active site. Moreover, DiCDD and TCDD increased the CYP1B1 gene expression and catalytic activity in porcine granulosa cells. On the other hand, TCDD demonstrated higher than DiCDD calculated affinity to pig CYP1B1, hindering TCDD exit from the active site. The great distance between CYP1B1's heme and TCDD also might contribute to the lower hydroxylation effectiveness of TCDD compared to that of DiCDD. Moreover, the narrow active site of pig CYP1B1 may immobilize TCDD molecule, inhibiting its hydroxylation. The results of the access channel analysis and the distance from pig CYP1B1's heme to TCDD suggest that the metabolizing potential of pig CYP1B1 is higher than that of pig CYP1A1. However, this potential is probably not sufficiently high to considerably improve the slow TCDD biodegradation.


Assuntos
Citocromo P-450 CYP1B1/metabolismo , Dioxinas/metabolismo , Suínos/metabolismo , Animais , Biocatálise , Citocromo P-450 CYP1B1/química , Dioxinas/química , Modelos Moleculares , Estrutura Molecular
3.
Bioorg Med Chem ; 27(2): 285-304, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30553624

RESUMO

Selective cytochrome P450 (CYP) 1B1 inhibition has potential as an anticancer strategy that is unrepresented in the current clinical arena. For development of a selective inhibitor, we focused on the complexity caused by sp3-hybridized carbons and synthesized a series of benzo[h]chromone derivatives linked to a non-aromatic B-ring using α-naphthoflavone (ANF) as the lead compound. Ring structure comparison suggested compound 37 as a suitable cyclohexyl-core with improved solubility. Structural evolution of 37 produced the azide-containing cis-49a, which had good properties in three important respects: (1) selectivity for CYP1B1 over CYP1A1 and CYP1A2 (120-times and 150-times, respectively), (2) greater inhibitory potency of >2 times that of ANF, and (3) improved solubility. The corresponding aromatic B-ring compound 59a showed low selectivity and poor solubility. To elucidate the binding mode, we performed X-ray crystal structure analysis, which revealed the interaction mode and explained the subtype selectivity of cis-49a.


Assuntos
Benzoflavonas/química , Inibidores do Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1B1/antagonistas & inibidores , Benzoflavonas/síntese química , Domínio Catalítico , Cristalografia por Raios X , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A2/química , Inibidores do Citocromo P-450 CYP1A2/síntese química , Citocromo P-450 CYP1B1/química , Desenho de Drogas , Escherichia coli/genética , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Solubilidade , Relação Estrutura-Atividade
4.
J Med Chem ; 61(23): 10901-10909, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30422652

RESUMO

Cytochrome P450 1B1 (CYP1B1) was found to be universally expressed in various tumors. Herein, we reported near-infrared fluorescent imaging probes for tumor detection via visualizing CYP1B1. After introducing the linker to a CYP1B1 selective inhibitor we found previously, we got the resulting compound 5b which kept strong inhibition ability against CYP1B1 (IC50 = 8.7 ± 1.2 nM) and high selectivity. Then, in vitro microscope studies and cell binding assay of probes indicated that the corresponding probe 6b could specifically be accumulated in CYP1B1 overexpressed colorectal cancer cell HCT-15 and showed satisfying binding affinity to target. During the in vivo noninvasive optical imaging, 6b was proved to rapidly lighten tumor in vivo as early as 6 h after injection. This work is the first attempt to visualize CYP1B1 for noninvasive imaging of tumor which could provide new approach for tumor diagnosis.


Assuntos
Neoplasias Colorretais/diagnóstico por imagem , Citocromo P-450 CYP1B1/metabolismo , Desenho de Drogas , Microscopia de Fluorescência , Sondas Moleculares/síntese química , Sondas Moleculares/metabolismo , Animais , Linhagem Celular Tumoral , Técnicas de Química Sintética , Neoplasias Colorretais/patologia , Citocromo P-450 CYP1B1/química , Humanos , Camundongos , Modelos Moleculares , Sondas Moleculares/farmacocinética , Conformação Proteica , Distribuição Tecidual
5.
J Med Chem ; 61(20): 9229-9245, 2018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30216063

RESUMO

Cytochrome P450 (CYP) 1B1 is involved in the bioactivation of procarcinogens and drug resistance. To obtain selective CYP1B1 inhibitors over CYP1A1, we synthesized four series of estrane derivatives: (1) 12 estrone (E1)- and 17ß-estradiol (E2)-derivatives bearing a 3- or a 4-pyridinyl core at C2, C3, or C4, (2) eight estrane derivatives with different sulfur groups at C3, (3) 19 E1- and E2-derivatives bearing distinct aryls at C2, and (4) five D-ring derivatives. E2-derivatives were more active than oxidized E1-analogues, thus highlighting the key role of 17ß-OH for interaction with CYP1B1. 2-(4-Fluorophenyl)-E2 was the best CYP1B1 inhibitor (IC50 = 0.24 µM), with a selectivity index (SI) of 20 over CYP1A1. Furthermore, the addition of a C17α-ethynyl group as D-ring modification improved the selectivity index to 25 with only a slight loss of activity (IC50 = 0.37 µM). Our docking results showed that these compounds fit better into the CYP1B1 binding site than that of CYP1A1.


Assuntos
Citocromo P-450 CYP1B1/antagonistas & inibidores , Inibidores das Enzimas do Citocromo P-450/síntese química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Desenho de Drogas , Estranos/síntese química , Estranos/farmacologia , Técnicas de Química Sintética , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/metabolismo , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/metabolismo , Estranos/química , Estranos/metabolismo , Simulação de Acoplamento Molecular , Conformação Proteica , Enxofre/química
6.
Biosci Rep ; 38(4)2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-29903728

RESUMO

Primary congenital glaucoma (PCG) is an inherited blinding eye disease. The CYP1B1 gene was identified as a causal gene for PCG, and many mutations have been found, but no studies have focussed on the molecular epidemiology of CYP1B1 in Chinese populations. We aimed to explore the CYP1B1 mutation hotspots in Chinese PCG patients and the possible impact of these mutations on the protein structure and function. First, we performed a meta-analysis on seven datasets of Chinese populations and found L107V and R390H to be the most common CYP1B1 mutations with allele frequencies of 3.19% and 3.09%, respectively. Then, a series of bioinformatics tools were applied to determine the sequence conservative properties, model the 3D structures, and study the dynamics changes. L107 and R390 are highly conserved residues in close proximity to the hemoglobin-binding region and the active site cavity (ASC), respectively. The mutations changed the distribution of hydrogen bonds and the local electrostatic potential. Long-term molecular dynamics (MD) simulations demonstrated the destabilization of the mutant proteins, especially at the ASC, whose solvent-accessible surface areas (SASAs) were significantly decreased. Compared with the wild-type (WT) protein, the overall structures of the mutants are associated with subtle but significant changes, and the ASC seems to adopt such structures that are not able to perform the WT-like functionality. Therefore, L107V and R390H might be the most important pathogenic mutations in Chinese PCG patients.


Assuntos
Citocromo P-450 CYP1B1/genética , Glaucoma/congênito , Glaucoma/genética , Mutação , Polimorfismo de Nucleotídeo Único , Sequência de Aminoácidos , Grupo com Ancestrais do Continente Asiático/genética , Sequência de Bases , China/epidemiologia , Biologia Computacional , Citocromo P-450 CYP1B1/química , Feminino , Deleção de Genes , Glaucoma/epidemiologia , Humanos , Mutação INDEL , Masculino , Modelos Moleculares , Mutagênese Insercional , Mutação de Sentido Incorreto , Conformação Proteica
7.
Artigo em Inglês | MEDLINE | ID: mdl-29763690

RESUMO

The environmental polycyclic aromatic hydrocarbons (PAH) and dioxins are carcinogens and their adverse effects have been largely attributed to the activation of AhR. Hesperetin is a flavonone found abundantly in citrus fruits and has been shown to be a biologically active agent. In the present study, the effect of hesperetin on the nuclear translocation of AhR and the downstream gene expression was investigated in MCF-7 cells. Confocal microscopy indicated that 7, 12-dimethylbenz[α]anthracene (DMBA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) -induced nuclear translocation of AhR was deterred by hesperetin treatment. The reduced nuclear translocation could also be observed in Western analysis. Reporter-gene assay further illustrated that the induced XRE transactivation was weakened by the treatment of hesperetin. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay demonstrated that the gene expressions of CYP1A1, 1A2, and 1B1 followed the same pattern of AhR translocation. These results suggested that hesperetin counteracted AhR transactivation and suppressed the downstream gene expression.


Assuntos
Antineoplásicos Fitogênicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Hesperidina/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , 9,10-Dimetil-1,2-benzantraceno/antagonistas & inibidores , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/patologia , Neoplasias da Mama/prevenção & controle , Carcinógenos Ambientais/química , Carcinógenos Ambientais/toxicidade , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1/antagonistas & inibidores , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Suplementos Nutricionais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter/efeitos dos fármacos , Humanos , Células MCF-7 , Microscopia Confocal , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Dibenzodioxinas Policloradas/antagonistas & inibidores , Dibenzodioxinas Policloradas/química , Receptores de Hidrocarboneto Arílico/metabolismo
8.
Sci Rep ; 8(1): 4498, 2018 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-29540704

RESUMO

Juvenile onset open-angle glaucoma (JOAG) affects patients before 40 years of age, causing high intraocular pressure and severe optic nerve damage. To expand the mutation spectrum of the causative genes in JOAG, with a view to identify novel disease-causing mutations, we investigated MYOC, OPTN, NTF4, WDR36 and CYP1B1 in a cohort of 67 unrelated Chinese JOAG patients. Whole exome sequencing was used to identify possible pathogenic mutations, which were further excluded in normal controls. After sequencing and the use of a database pipeline, as well as predictive assessment filtering, we identified a total of six mutations in three genes, MYOC, OPTN and CYP1B1. Among them, 2 heterozygous mutations in MYOC (c. 1109C > T, p. (P370L); c. 1150G > C, p. (D384H)), 2 heterozygous mutations in OPTN (c. 985A > G, p.(R329G); c. 1481T > G, p. (L494W)) and 2 homozygous mutations in CYP1B1 (c. 1412T > G, p.(I471S); c. 1169G > A, p.(R390H)) were identified as potentially causative mutations. No mutation was detected in NTF4 or WDR36. Our results enrich the mutation spectra and frequencies of MYOC, OPTN and CYP1B1 in JOAG among the Chinese population. Further studies are needed to address the pathogenicity of each of the mutations detected in this study.


Assuntos
Citocromo P-450 CYP1B1/genética , Proteínas do Citoesqueleto/genética , Proteínas do Olho/genética , Predisposição Genética para Doença , Glaucoma de Ângulo Aberto/genética , Glicoproteínas/genética , Mutação , Fator de Transcrição TFIIIA/genética , Adulto , Idade de Início , Alelos , Sequência de Aminoácidos , Grupo com Ancestrais do Continente Asiático/genética , China , Citocromo P-450 CYP1B1/química , Proteínas do Citoesqueleto/química , Proteínas do Olho/química , Feminino , Estudos de Associação Genética , Genótipo , Glaucoma de Ângulo Aberto/epidemiologia , Glicoproteínas/química , Humanos , Masculino , Conformação Proteica , Relação Estrutura-Atividade , Fator de Transcrição TFIIIA/química , Sequenciamento Completo do Exoma , Adulto Jovem
9.
Xenobiotica ; 48(6): 565-575, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28648140

RESUMO

1. 1-Chloropyrene, one of the major chlorinated polycyclic aromatic hydrocarbon contaminants, was incubated with human cytochrome P450 (P450 or CYP) enzymes including CYP1A1, 1A2, 1B1, 2A6, 2A13, 2B6, 2C9, 2D6, 2E1, 3A4 and 3A5. Catalytic differences in 1-chloropyrene oxidation by polymorphic two CYP1B1 and five CYP2A13 allelic variants were also examined. 2. CYP1A1 oxidized 1-chloropyrene at the 6- and 8-positions more actively than at the 3-position, while both CYP1B1.1 and 1B1.3 preferentially catalyzed 6-hydroxylation. 3. Five CYP2A13 allelic variants oxidized 8-hydroxylation much more than 6- and 3-hydroxylation, and the variant CYP2A13.3 was found to slowly catalyze these reactions with a lower kcat value than other CYP2A13.1 variants. 4. CYP2A6 catalyzed 1-chloropyrene 6-hydroxylation at a higher rate than the CYP2A13 enzymes, but the rate was lower than the CYP1A1 and 1B1 variants. Other human P450 enzymes had low activities towards 1-chloropyrene. 5. Molecular docking analysis suggested differences in the interaction of 1-chloropyrene with active sites of CYP1 and 2 A enzymes. In addition, a naturally occurring Thr134 insertion in CYP2A13.3 was found to affect the orientation of Asn297 in the I-helix in interacting with 1-chloropyrene (and also 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK) and caused changes in the active site of CYP2A13.3 as compared with CYP2A13.1.


Assuntos
Alelos , Hidrocarboneto de Aril Hidroxilases , Citocromo P-450 CYP1B1 , Simulação de Acoplamento Molecular , Pirenos/química , Hidrocarboneto de Aril Hidroxilases/química , Hidrocarboneto de Aril Hidroxilases/genética , Biocatálise , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/genética , Humanos , Oxirredução
10.
Environ Toxicol Pharmacol ; 55: 175-185, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28886471

RESUMO

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD; polycyclic aromatic hydrocarbon) is a persistent and ubiquitous environmental contaminant that causes a wide variety of deleterious effects. In this study, the DNA damage and apoptotic activity induced by TCDD was examined using in silico and in vitro approaches. In silico study showed that conformational changes and energies involved in the binding of TCDD to cytochrome P450 1B1 (CYP1B1) were crucial for its target proteins. Moreover, activated TCDD had high affinity to bind with aryl hydrocarbon receptor (AhR), with a binding energy of -564.7 Kcal/mol. Further, TCDD-CYP1B1 complex showed strong binding affinity for caspase 3, showing a binding energy of -518.5 Kcal/mol, and the docking of caspase inhibitors in the complex showed weak interaction with low binding energy as compared to TCDD-CYP1B1 caspase complexes. Interestingly, TCDD-induced apoptosis was significantly suppressed in Ac-DEVD-CMK-pretreated cells. The DNA damage activity of TCDD was quantified by comet tail formation and γ-H2AX foci formation in HaCaT cells. The role of CYP1B1 and AhR in DNA damage and apoptosis was demonstrated, and clotrimazole as well as knockdown of CYP1B1 and AhR could inhibit TCDD activation and suppress DNA damage followed by apoptosis in HaCaT cells. Moreover, TCDD increased expression of p53 and PUMA and our data showed that TCDD induced DNA damage followed by p53-mediated apoptosis. This study highlights the critical role of CYP1B1 and AhR in TCDD activity and proposes that inhibition of these key molecules might serve as a potential therapeutic approach for treatment of allergy and cancer.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Dano ao DNA , Dibenzodioxinas Policloradas/efeitos adversos , Receptores de Hidrocarboneto Arílico/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose , Linhagem Celular , Simulação por Computador , Citocromo P-450 CYP1B1/química , Exposição Ambiental/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Dibenzodioxinas Policloradas/farmacologia , Conformação Proteica/efeitos dos fármacos
11.
Chem Biol Drug Des ; 90(6): 1226-1236, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28632937

RESUMO

Cytochromes P450 family 1 (CYP1) are responsible for the metabolism of procarcinogens, for example polycyclic aromatic hydrocarbons and aromatic and heterocyclic amines. The inhibition of CYP1 activity is examined in terms of chemoprevention and cancer chemotherapy. We designed and synthesized a series of trans-stilbene derivatives possessing a combination of methoxy and methylthio functional groups attached in different positions to the trans-stilbene skeleton. We determined the effects of synthesized compounds on the activities of human recombinant CYP1A1, CYP1A2 and CYP1B1 and, to explain the variation of inhibitory potency of methoxystilbene derivatives and their methylthio analogues, we employed computational analysis. The compounds were docked to CYP1A1, CYP1A2 and CYP1B1 binding sites with the use of Accelrys Discovery Studio 4.0 by the CDOCKER procedure. For CYP1A2 and CYP1B1, values of scoring functions correlated well with inhibitory potency of stilbene derivatives. All compounds were relatively poor inhibitors of CYP1A2 that possess the most narrow and flat enzyme cavity among CYP1s. For the most active CYP1A1 inhibitor, 2-methoxy-4'-methylthio-trans-stilbene, a high number of molecular interactions was observed, although the interaction energies were not distinctive.


Assuntos
Família 1 do Citocromo P450/metabolismo , Inibidores Enzimáticos/metabolismo , Estilbenos/metabolismo , Sítios de Ligação , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Família 1 do Citocromo P450/química , Família 1 do Citocromo P450/genética , Inibidores Enzimáticos/síntese química , Humanos , Isomerismo , Espectroscopia de Ressonância Magnética , Conformação Molecular , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Estilbenos/química , Termodinâmica
12.
Molecules ; 22(2)2017 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-28212337

RESUMO

In this study, a series of regioisomeric acetylenic sulfamoylquinolines are designed, synthesized, and tested in vitro for their antiproliferative activity against three human breast cacer cell lines (T47D, MCF-7, and MDA-MB-231) and a human normal fibroblast (HFF-1) by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay. The antiproliferative activity of the tested acetylenic quinolinesulfonamides is comparable to that of cisplatin. The bioassay results demonstrate that most of the tested compounds show potent antitumor activities, and that some compounds exhibit better effects than the positive control cisplatin against various cancer cell lines. Among these compounds, 4-(3-propynylthio)-7-[N-methyl-N-(3-propynyl)sulfamoyl]quinoline shows significant antiprolierative activity against T47D cells with IC50 values of 0.07 µM. In addition, 2-(3-Propynylthio)-6-[N-methyl-N-(3-propynyl)sulfa-moyl]quinoline and 2-(3-propynylseleno)-6-[N-methyl-N-(3-propynyl)sulfamoyl]quinoline display highly effective atitumor activity against MDA-MB-231 cells, with IC50 values of 0.09 and 0.50 µM, respectively. Furthermore, most of the tested compounds show a weak cytotoxic effect against the normal HFF-1 cell line. Additionally, in order to suggest a mechanism of action for their activity, all compounds are docked into the binding site of two human cytochrome P450 (CYP) isoenzymes. These data indicate that some of the title compounds display significant cytotoxic activity, possibly targeting the CYPs pathways.


Assuntos
Alquinos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Simulação de Acoplamento Molecular , Quinolinas/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Antineoplásicos/síntese química , Sítios de Ligação , Domínio Catalítico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/metabolismo , Relação Dose-Resposta a Droga , Humanos , Ligações de Hidrogênio , Conformação Molecular , Ligação Proteica , Relação Estrutura-Atividade , Sulfonamidas/síntese química
13.
Clin Genet ; 90(4): 378-82, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27272408

RESUMO

The genetic basis of congenital glaucoma with systemic anomalies is largely unknown. Whole exome sequencing (WES) in 10 probands with congenital glaucoma and variable systemic anomalies identified pathogenic or likely pathogenic variants in three probands; in two of these, a combination of two Mendelian disorders was found to completely explain the patients' features whereas in the third case only the ocular findings could be explained by the genetic diagnosis. The molecular diagnosis for glaucoma included two cases with compound heterozygous or homozygous pathogenic alleles in CYP1B1 and one family with a dominant pathogenic variant in FOXC1; the second genetic diagnosis for the additional systemic features included compound heterozygous mutations in NPHS1 in one family and a heterozygous 18q23 deletion in another pedigree. These findings show the power of WES in the analysis of complex conditions and emphasize the importance of CYP1B1 screening in patients with congenital glaucoma regardless of the presence/absence of other systemic anomalies.


Assuntos
Glaucoma/genética , Alelos , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/genética , Análise Mutacional de DNA , Exoma , Feminino , Fatores de Transcrição Forkhead/química , Fatores de Transcrição Forkhead/genética , Glaucoma/congênito , Heterozigoto , Humanos , Lactente , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Linhagem , Análise de Sequência de DNA
14.
PLoS One ; 11(5): e0156252, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27243976

RESUMO

Glaucoma, the leading cause of irreversible blindness, appears in various forms. Mutations in CYP1B1 result in primary congenital glaucoma (PCG) by an autosomal recessive mode of inheritance while it acts as a modifier locus for primary open angle glaucoma (POAG). We investigated the molecular basis of the variable phenotypes resulting from the defects in CYP1B1 by using subclones of 23 CYP1B1 mutants reported in glaucoma patients, in a cell based system by measuring the dual activity of the enzyme to metabolize both retinol and 17ß-estradiol. Most variants linked to POAG showed low steroid metabolism while null or very high retinol metabolism was observed in variants identified in PCG. We examined the translational turnover rates of mutant proteins after the addition of cycloheximide and observed that the levels of enzyme activity mostly corroborated the translational turnover rate. We performed extensive normal mode analysis and molecular-dynamics-simulations-based structural analyses and observed significant variation of fluctuation in certain segmental parts of the mutant proteins, especially at the B-C and F-G loops, which were previously shown to affect the dynamic behavior and ligand entry/exit properties of the cytochrome P450 family of proteins. Our molecular study corroborates the structural analysis, and suggests that the pathologic state of the carrier of CYP1B1 mutations is determined by the allelic state of the gene. To our knowledge, this is the first attempt to dissect biological activities of CYP1B1 for correlation with congenital and adult onset glaucomas.


Assuntos
Citocromo P-450 CYP1B1/genética , Glaucoma/enzimologia , Glaucoma/genética , Proteínas Mutantes/genética , Adulto , Sequência de Aminoácidos , Arginina/química , Linhagem Celular , Sequência Conservada , Citocromo P-450 CYP1B1/química , Citocromo P-450 CYP1B1/metabolismo , Estradiol/metabolismo , Estudos de Associação Genética , Variação Genética , Glaucoma/congênito , Células HEK293 , Humanos , Recém-Nascido , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vitamina A/metabolismo
15.
J Mol Recognit ; 29(8): 370-90, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26916064

RESUMO

Recent trends in new drug discovery of anticancer drugs have made oncologists more aware of the fact that the new drug discovery must target the developing mechanism of tumorigenesis to improve the therapeutic efficacy of antineoplastic drugs. The drugs designed are expected to have high affinity towards the novel targets selectively. Current research highlights overexpression of CYP450s, particularly cytochrome P450 1A1 (CYP1A1), in tumour cells, representing a novel target for anticancer therapy. However, the CYP1 family is identified as posing significant problems in selectivity of anticancer molecules towards CYP1A1. Three members have been identified in the human CYP1 family: CYP1A1, CYP1A2 and CYP1B1. Although sequences of the three isoform have high sequence identity, they have distinct substrate specificities. The understanding of macromolecular features that govern substrate specificity is required to understand the interplay between the protein function and dynamics, design novel antitumour compounds that could be specifically metabolized by only CYP1A1 to mediate their antitumour activity and elucidate the reasons for differences in substrate specificity profile among the three proteins. In the present study, we employed a combination of computational methodologies: molecular docking and molecular dynamics simulations. We utilized eight substrates for elucidating the difference in substrate specificity of the three isoforms. Lastly, we conclude that the substrate specificity of a particular substrate depends upon the type of the active site residues, the dynamic motions in the protein structure upon ligand binding and the physico-chemical characteristics of a particular ligand. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Antineoplásicos/farmacologia , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1B1/química , Antineoplásicos/química , Domínio Catalítico/efeitos dos fármacos , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Bases de Dados de Compostos Químicos , Desenho de Drogas , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Especificidade por Substrato
16.
Mol Divers ; 18(4): 865-78, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25028215

RESUMO

Recently, CYP1 enzymes are documented for selective metabolism of anticancer leads in cancer prevention and/or progression. Elucidation of specificity of substrates/inhibitors of CYP1 isoforms plays a vital role in design of more selective and potent anticancer leads. However, an area of concern is the broad range of substrate specificities and planar nature of substrates with limited dataset which makes it difficult to predict their site of metabolism (SOM) accurately. In the present study, various models for prediction of site of metabolism in case of CYP1A1, CYP1A2, and CYP1B1 substrates were developed using MetaSite, molecular docking, and quantum chemical descriptors. The predictive accuracy of MetaSite, molecular docking, and quantum chemical descriptors in identifying experimental site of metabolism was analyzed at three levels; top rank, top three ranks, and top five ranks. Two quantum chemical descriptors, chemical hardness and local nucleophilicity are proposed for the prediction of CYP-mediated SOM for the first time. The predictive accuracy shown by chemical hardness at top three ranks was 83.3, 85.7, and 84.6 % for CYP1A1, CYP1A2 and CYP1B1, respectively, whereas local nucleophilicity gave poor predictions of 50, 42.8, and 46.2 %, respectively. The predictability of chemical hardness descriptor outperformed at all three levels of ranks for CYP1A1, CYP1A2, and CYP1B1. Hence, we propose chemical hardness as an useful quantum chemical descriptor for prediction of metabolically vulnerable prints in CYP1A1, CYP1A2, and CYP1B1 mediated metabolism and support the optimization efforts in drug discovery and development programs.


Assuntos
Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A2/química , Citocromo P-450 CYP1B1/química , Modelos Químicos , Modelos Moleculares , Sítios de Ligação , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Teoria Quântica , Reprodutibilidade dos Testes , Especificidade por Substrato
17.
Ann Hum Genet ; 78(4): 255-63, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24942078

RESUMO

Primary congenital glaucoma (PCG) is responsible for a significant proportion of childhood blindness in Tunisia. Early prevention based on genetic diagnosis is therefore required. This study sought to determine the frequency of CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1) mutations in 18 PCG patients, recruited from Central and Southern of Tunisia. Genomic DNA was extracted and the coding regions of CYP1B1 were analysed by direct sequencing. A phylogenetic network of CYP1B1 haplotypes was drawn using the median-joining algorithm. Sequence analysis revealed a "tetra-allelic mutation" (two novel mutations, p.F231I and p.P437A in the homozygous state) in one patient. The healthy members of his family carried those variations on the same allele. Two previously described mutations p.G61E and c.535delG were also identified in the homozygous state in seven and two probands, respectively. Seven single-nucleotide polymorphisms were identified and used to generate haplotypes. Our results showed that the CYP1B1 mutations were present in 55% of Tunisian PCG patients' alleles. Haplotype analysis allowed us to define the proto-haplotype and to confirm historical migratory flows. Establishment of PCG genetic aetiology in Tunisia will improve genetic diagnosis and counselling.


Assuntos
Citocromo P-450 CYP1B1/genética , Glaucoma/congênito , Glaucoma/genética , Mutação , Consanguinidade , Citocromo P-450 CYP1B1/química , Análise Mutacional de DNA , Feminino , Genótipo , Glaucoma/diagnóstico , Haplótipos , Humanos , Lactente , Recém-Nascido , Masculino , Modelos Moleculares , Linhagem , Filogenia , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Tunísia
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