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3.
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
4.
Eur J Med Chem ; 165: 115-132, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30665142

RESUMO

Inhibition of cyclin dependent kinase 4 (Cdk4) prevents cancer cells from entering the early G0/G1 phase of the cell division cycle whereas inhibiting tubulin polymerization blocks cancer cells' ability to undergo mitosis (M) late in the cell cycle. We had reported earlier that two non-planar and relatively non-toxic fascaplysin derivatives, an indole and a tryptoline, inhibit Cdk4 with IC50 values of 6.2 and 10 µM, respectively. Serendipitously, we had also found that they inhibited tubulin polymerization. The molecules were efficacious in mouse tumor models. We have now identified Cink4T in a 59-compound quinazolinone library, designed on the basis of ligand-based virtual screening, as a compound that inhibits Cdk4 and tubulin. Its IC50 value for Cdk4 inhibition is 0.47 µM and >50 µM for inhibition of Cdk1, Cdk2, Cdk6, Cdk9. Cink4T inhibits tubulin polymerization with an IC50 of 0.6 µM. Molecular modelling studies on Cink4T with Cdk4 and tubulin crystal structures lend support to these observations. Cancer cell cycle analyses confirm that Cink4T blocks cells at both G0/G1 and M phases as it should if it were to inhibit both Cdk4 and tubulin polymerization. Our results show, for the very first time, that virtual screening can be used to design novel inhibitors that can potently block two crucial phases of the cell division cycle.


Assuntos
Antineoplásicos/química , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinazolinonas/farmacologia , Tubulina (Proteína)/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Polimerização/efeitos dos fármacos , Quinazolinonas/química , Bibliotecas de Moléculas Pequenas , Tubulina (Proteína)/metabolismo
5.
Bioorg Chem ; 85: 152-158, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30612081

RESUMO

Aggregation/misfolding of α-synuclein and ßA4 proteins cause neuronal cell death (NCD) associated with Parkinson's and Alzheimer's disease. It has been suggested that a heat shock protein-90 (Hsp90) inhibitor can prevent NCD by activating the heat shock transcription factor-1 which, in turn, upregulates molecular chaperones such as Hsp70 that targets aggregated/misfolded proteins for refolding/degradation. We have isolated radicicol, an Hsp90 inhibitor, from a fungus occurring in the crevices of marble rocks of Central India. Radicicol, which was found to be a strong antioxidant, was tested for its ability to rescue yeast cells from death induced by expression of wild-type α-synuclein, its more toxic A53T mutant, and ßA4. It effectively overcomes wild-type/mutant α-synuclein mediated yeast cell death, concomitantly diminishes ROS levels, reverses mitochondrial dysfunction and prevents nuclear DNA-fragmentation, a hallmark of apoptosis. Surprisingly however, radicicol is unable to rescue yeast cells from death triggered by expression of secreted ßA4. Moreover, although radicicol acts as an antioxidant it fails to prevent yeast cell death inflicted by the proapoptotic protein, Bax. Our results indicate that radicicol specifically targets aggregated/misfolded α-synuclein's toxicity and opens up the possibility of using multiple yeast assays to screen natural product libraries for compounds that would unambiguously target α-synuclein aggregation/misfolding.

6.
Eur J Pharm Sci ; 128: 118-127, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30502452

RESUMO

Aggregated Aß peptides which cause amyloid deposits, a characteristic of Alzheimer's disease (AD), activate a stress response in the endoplasmic reticulum (ER), known as the unfolded protein response, UPRER. Nascent UPRER induction helps in reducing ER stress by eliminating accumulated misfolded/aggregated secretory proteins. However, prolonged UPRER induction may trigger apoptosis. Here we show that, when expressed in yeast with an NH2-terminal secretory signal sequence (ss), the 42-amino acid human Aß42 (h_Aß42), but not the mouse/ratAß42 (m_Aß42) which reportedly does not misfold/aggregate, induces UPRER as monitored via an eGFP reporter. We also show that expression of ss-h_Aß42, not ss-m_Aß42, blocks yeast cell growth, with cells expressing ss-h_Aß42 manifesting distinctive features of apoptosis such as loss of mitochondrial membrane potential, increase in ROS levels and DNA fragmentation. Screening for suppressors of ss-h_Aß42-activated UPRER-eGFP induction, in a computationally-designed 29-compound methoxy-stilbene library, revealed three compounds that reduce >95% of UPRER-eGFP induction at 5 µM concentration, with EC50 values of 40-50 nM. Surprisingly, the compounds also rescue yeast cells from ss-h_Aß42-mediated apoptosis, with EC50-s of 50-60 nM. These results provide direct evidence, probably for the first time, that there is a direct correlation between deactivation of UPRER and attenuation of apoptosis.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Apoptose/fisiologia , Fragmentos de Peptídeos/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Estilbenos/farmacologia , Peptídeos beta-Amiloides/metabolismo , Animais , Retículo Endoplasmático , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Camundongos , Estrutura Molecular , Fragmentos de Peptídeos/metabolismo , Ratos , Espécies Reativas de Oxigênio , Saccharomyces cerevisiae/metabolismo , Estilbenos/síntese química , Estilbenos/química , Relação Estrutura-Atividade , Resposta a Proteínas não Dobradas
7.
Bioorg Med Chem Lett ; 29(3): 454-460, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30579794

RESUMO

Herein, we have identified yeast Sec22p (ySec22p), a SNARE protein essential for endoplasmic reticulum to Golgi trafficking, as a suppressor of Bax-induced yeast apoptosis and corroborated published observations that ySec22p suppresses α-synuclein's toxicity in yeast. It has been suggested that compounds which enhance expression, in neurons, of human homologues of ySec22p (Sec22Bp/Sec22p/Sec22A) would prevent synucleinopathies, such as Parkinson's disease. With the aim of finding a small molecule that would mimic ySec22p, a library of natural products consisting of 394-compounds was screened using yeast cells that express either human α-synuclein or human Bax. The antioxidant aegeline, an alkaloid-amide occurring in the leaves of the plant Aegle marmelos Correa, was the only molecule that overcame apoptosis induced by both α-synuclein and Bax in yeast. Besides, aegeline also prevented growth block in cells expressing the more toxic A53T α-synuclein mutant. Restoration of cell growth occurred through inhibition of increased ROS levels, mitochondrial membrane potential loss and nuclear DNA fragmentation, characteristics of apoptosis manifested in α-synuclein or Bax-expressing cells. These results highlight the importance of yeast systems to identify rapidly molecules that may prevent the onset of apoptosis that occurs in Parkinson's disease.


Assuntos
Aegle/química , Amidas/farmacologia , Antioxidantes/farmacologia , Produtos Biológicos/farmacologia , Proteínas R-SNARE/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Amidas/química , Amidas/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Apoptose/efeitos dos fármacos , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Relação Dose-Resposta a Droga , Estrutura Molecular , Proteínas R-SNARE/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , alfa-Sinucleína/metabolismo , Proteína X Associada a bcl-2/metabolismo
8.
Bioorg Med Chem ; 26(23-24): 6076-6086, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30448188

RESUMO

Expression of cytochrome P450-1A1 (CYP1A1) is suppressed under physiologic conditions but is induced (a) by polycyclic aromatic hydrocarbons (PAHs) which can be metabolized by CYP1A1 to carcinogens, and (b) in majority of breast cancers. Hence, phytochemicals or dietary flavonoids, if identified as CYP1A1 inhibitors, may help in preventing PAH-mediated carcinogenesis and breast cancer. Herein, we have investigated the cancer chemopreventive potential of a flavonoid-rich Indian medicinal plant, Pongamia pinnata (L.) Pierre. Methanolic extract of its seeds inhibits CYP1A1 in CYP1A1-overexpressing normal human HEK293 cells, with IC50 of 0.6 µg/mL. Its secondary metabolites, the furanoflavonoids pongapin/lanceolatin B, inhibit CYP1A1 with IC50 of 20 nM. Although the furanochalcone pongamol inhibits CYP1A1 with IC50 of only 4.4 µM, a semisynthetic pyrazole-derivative P5b, has ∼10-fold improved potency (IC50, 0.49 µM). Pongapin/lanceolatin B and the methanolic extract of P. pinnata seeds protect CYP1A1-overexpressing HEK293 cells from B[a]P-mediated toxicity. Remarkably, they also block the cell cycle of CYP1A1-overexpressing MCF-7 breast cancer cells, at the G0-G1 phase, repress cyclin D1 levels and induce cellular-senescence. Molecular modeling studies demonstrate the interaction pattern of pongapin/lanceolatin B with CYP1A1. The results strongly indicate the potential of methanolic seed-extract and pongapin/lanceolatin B for further development as cancer chemopreventive agents.


Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Citocromo P-450 CYP1A1/antagonistas & inibidores , Flavonas/farmacologia , Furanos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Benzopiranos/síntese química , Benzopiranos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Citocromo P-450 CYP1A1/biossíntese , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Flavonas/síntese química , Flavonas/química , Citometria de Fluxo , Furanos/síntese química , Furanos/química , Células HEK293 , Humanos , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade
9.
Medchemcomm ; 9(2): 371-382, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30108931

RESUMO

CYP1A1 is thought to mediate carcinogenesis in oral, lung and epithelial cancers. In order to identify a CYP1A1 inhibitor from an edible plant, 394 natural products in the IIIM's natural product repository were screened, at 10 µM concentration, using CYP1A1-Sacchrosomes™ (i.e. microsomal enzyme isolated from recombinant baker's yeast). Twenty-seven natural products were identified that inhibited 40-97% of CYP1A1's 7-ethoxyresorufin-O-deethylase activity. The IC50 values of the 'hits', belonging to different chemical scaffolds, were determined. Their selectivity was studied against a panel of 8 CYP-Sacchrosomes™. In order to assess cellular efficacy, the 'hits' were screened for their capability to inhibit CYP enzymes expressed within live recombinant human embryonic kidney (HEK293) cells from plasmids encoding specific CYP genes (1A2, 1B1, 2C9, 2C19, 2D6, 3A4). Isopimpinellin (IN-475; IC50, 20 nM) and karanjin (IN-195; IC50, 30 nM) showed the most potent inhibition of CYP1A1 in human cells. Isopimpinellin is found in celery, parsnip, fruits and in the rind and pulp of limes whereas different parts of the Indian beech tree, which contain karanjin, have been used in traditional medicine. Both isopimpinellin and karanjin negate the cellular toxicity of CYP1A1-mediated benzo[a]pyrene. Molecular docking and molecular dynamic simulations with CYP isoforms rationalize the observed trends in the potency and selectivity of isopimpinellin and karanjin.

10.
ACS Omega ; 3(8): 8553-8566, 2018 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31458985

RESUMO

Cytochrome P450 family 1 (CYP1) enzymes catalyze the metabolic activation of environmental procarcinogens such as benzo[a]pyrene, B[a]P, into carcinogens, which initiates the process of carcinogenesis. Thus, stopping the metabolic activation of procarcinogens can possibly prevent the onset of cancer. Several natural products have been reported to show unique ability in inhibiting CYP1 enzymes. We found that khellin, a naturally occurring furanochromone from Ammi visnaga, inhibits CYP1A1 enzyme with an IC50 value of 4.02 µM in CYP1A1-overexpressing human HEK293 suspension cells. To further explore this natural product for discovery of more potent and selective CYP1A1 inhibitors, two sets of semisynthetic derivatives were prepared. Treatment of khellin with alkali results in opening of a pyrone ring, yielding khellinone (2). Claisen-Schmidt condensation of khellinone (2) with various aldehydes in presence of potassium hydroxide, at room temperature, provides a series of furanochalcones 3a-v (khellinochalcones). Treatment of khellinone (2) with aryl aldehydes in the presence of piperidine, under reflux, affords the flavanone series of compounds 4a-p (khellinoflavanones). The khellinoflavanone 4l potently inhibited CYP1A1 with an IC50 value of 140 nM in live cells, with 170-fold selectivity over CYP1B1 (IC50 for CYP1B1 = 23.8 µM). Compound 4l at 3× IC50 concentration for inhibition of CYP1A1 completely protected HEK293 cells from CYP1A1-mediated B[a]P toxicity. Lung cancer cells, A549 (p53+) and Calu-1 (p53-null), blocked in growth at the S-phase by B[a]P were restored into the cell cycle by compound 4l. The results presented herein strongly indicate the potential of these khellin derivatives for further development as cancer chemopreventive agents.

11.
ACS Omega ; 3(8): 8903-8912, 2018 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31459022

RESUMO

CYP2D6, a cytochrome P450 (CYP) enzyme, metabolizes codeine to morphine. Within the human body, 0-15% of codeine undergoes O-demethylation by CYP2D6 to form morphine, a far stronger analgesic than codeine. Genetic polymorphisms in wild-type CYP2D6 (CYP2D6-wt) are known to cause poor-to-extensive metabolism of codeine and other CYP2D6 substrates. We have established a platform technology that allows stable expression of human CYP genes from chromosomal loci of baker's yeast cells. Four CYP2D6 alleles, (i) chemically synthesized CYP2D6.1, (ii) chemically synthesized CYP2D6-wt, (iii) chemically synthesized CYP2D6.10, and (iv) a novel CYP2D6.10 variant CYP2D6-C (i.e., CYP2D6.10A122V) isolated from a liver cDNA library, were cloned for chromosomal integration in yeast cells. When expressed in yeast, CYP2D6.10 enzyme shows weak activity compared with CYP2D6-wt and CYP2D6.1 which have moderate activity, as reported earlier. Surprisingly, however, the CYP2D6-C enzyme is far more active than CYP2D6.10. More surprisingly, although CYP2D6.10 is a known low metabolizer of codeine, yeast cells expressing CYP2D6-C transform >70% of codeine to morphine, which is more than twice that of cells expressing the extensive metabolizers, CYP2D6.1, and CYP2D6-wt. The latter two enzymes predominantly catalyze formation of codeine's N-demethylation product, norcodeine, with >55% yield. Molecular modeling studies explain the specificity of CYP2D6-C for O-demethylation, validating observed experimental results. The yeast-based CYP2D6 expression systems, described here, could find generic use in CYP2D6-mediated drug metabolism and also in high-yield chemical reactions that allow the formation of regio-specific dealkylation products.

12.
ACS Omega ; 3(8): 9513-9532, 2018 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-31459084

RESUMO

The overexpression of α-synuclein (α-syn) and its aggregation is the hallmark of Parkinson's disease. The α-syn aggregation results in the formation of Lewy bodies that causes neuronal cell death. Therefore, the small molecules that can protect neuronal cells from α-syn toxicity or inhibit the aggregation of α-syn could emerge as anti-Parkinson agents. Herein, a library of methoxy-stilbenes was screened for their ability to restore the cell growth from α-syn toxicity, using a yeast strain that stably expresses two copies of a chromosomally integrated human α-syn gene. Tetramethoxy-stilbene 4s, a nonantioxidant, was the most capable of restoring cell growth. It also rescues the more toxic cells that bear three copies of wild-type or A53T-mutant α-syn, from cell growth block. Its EC50 values for growth restoration of the 2-copy wild-type and the 3-copy mutant α-syn strains are 0.95 and 0.35 µM, respectively. Stilbene 4s mitigates mitochondrial membrane potential loss, negates ROS production, and prevents nuclear DNA-fragmentation, all hallmarks of apoptosis. However, 4s does not rescue cells from the death-inducing effects of Bax and ßA4, which suggest that 4s specifically inhibits α-syn-mediated toxicity in the yeast. Our results signify that simultaneous use of multiple yeast-cell-based screens can facilitate revelation of compounds that may have the potential for further investigation as anti-Parkinson's agents.

13.
Bioorg Med Chem Lett ; 27(24): 5409-5414, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29138024

RESUMO

The overexpression of CYP1 family of enzymes is reported to be associated with development of human carcinomas. It has been well reported that CYP1A1 specific inhibitors prevents carcinogenesis. Herein, thirteen pyridine-4-yl series of chalcones were synthesized and screened for inhibition of CYP1 isoforms 1A1, 1B1 and 1A2 in Sacchrosomes™ and live human HEK293 cells. The structure-activity relationship analysis indicated that chalcones bearing tri-alkoxy groups (8a and 8k) on non-heterocyclic ring displayed selective inhibition of CYP1A1 enzyme, with IC50 values of 58 and 65 nM, respectively. The 3,4,5-trimethoxy substituted derivative 8a have shown >10-fold selectivity towards CYP1A1 with respect to other enzymes of the CYP1 sub-family and >100-fold selectivity with respect to CYP2 and CYP3 family of enzymes. The potent and selective CYP1A1 inhibitor 8a displayed antagonism of B[a]P mediated activation of aromatic hydrocarbon receptor (AhR) in yeast cells, and also protected human cells from CYP1A1-mediated B[a]P toxicity in human cells. This potent and selective inhibitor of CYP1A1 enzyme have a potential for development as cancer chemopreventive agent.


Assuntos
Chalconas/química , Citocromo P-450 CYP1A1/antagonistas & inibidores , Propano/análogos & derivados , Sítios de Ligação , Chalconas/farmacologia , Cisplatino/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células HEK293 , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Propano/química , Propano/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Leveduras/efeitos dos fármacos , Leveduras/metabolismo
14.
Bioorg Med Chem Lett ; 27(24): 5400-5403, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29150398

RESUMO

The development of multi-drug resistance to existing anticancer drugs is one of the major challenges in cancer treatment. The over-expression of cytochrome P450 1B1 enzyme has been reported to cause resistance to cisplatin. With an objective to discover cisplatin-resistance reversal agents, herein, we report the evaluation of Glycyrrhiza glabra (licorice) extracts and its twelve chemical constituents for inhibition of CYP1B1 (and CYP1A1) enzyme in Sacchrosomes and live human cells. The hydroalcoholic extract showed potent inhibition of CYP1B1 in both Sacchrosomes as well as in live cells with IC50 values of 21 and 16 µg/mL, respectively. Amongst the total of 12 constituents tested, quercetin and glabrol showed inhibition of CYP1B1 in live cell assay with IC50 values of 2.2 and 15 µM, respectively. Both these natural products were found to be selective inhibitors of CYP1B1, and does not inhibit CYP2 and CYP3 family of enzymes (IC50 > 20 µM). The hydroalcoholic extract of G. glabra and quercetin (4) showed complete reversal of cisplatin resistance in CYP1B1 overexpressing triple negative MDA-MB-468 breast cancer cells. The selective inhibition of CYP1B1 by quercetin and glabrol over CYP2 and CYP3 family of enzymes was studied by molecular modeling studies.


Assuntos
Antineoplásicos/farmacologia , Citocromo P-450 CYP1B1/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/química , Glycyrrhiza/química , Extratos Vegetais/química , Quercetina/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Citocromo P-450 CYP1B1/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Glycyrrhiza/metabolismo , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Extratos Vegetais/farmacologia , Estrutura Terciária de Proteína , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
15.
J Agric Food Chem ; 65(34): 7440-7446, 2017 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-28782952

RESUMO

Naturally occurring polyphenolic compounds are of medicinal importance because of their unique antioxidant, anticancer, and chemopreventive properties. Baicalein, a naturally occurring polyhydroxy flavonoid possessing a diverse range of pharmacological activities, has been used in traditional medicines for treatment of various ailments. Apart from its isolation from natural sources, its synthesis has been reported via multistep chemical approaches. Here, we report a preparative-scale biotransformation, using whole yeast cells stably expressing human cytochrome P450 1A1 (CYP1A1) enzyme that allows regioselective C6-hydroxylation of 5,7-dihydroxyflavone (chrysin) to form 5,6,7-trihydroxyflavone (baicalein). Molecular modeling reveals why chrysin undergoes such specific hydroxylation mediated by CYP1A1. More than 92% reaction completion was obtained using a shake-flask based process that mimics fed-batch fermentation. Such highly efficient selective hydroxylation, using recombinant yeast cells, has not been reported earlier. Similar CYP-expressing yeast cell based systems are likely to have wider applications in the syntheses of medicinally important polyphenolic compounds.


Assuntos
Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/metabolismo , Flavanonas/metabolismo , Flavonoides/metabolismo , Saccharomyces cerevisiae/genética , Biocatálise , Biotransformação , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/isolamento & purificação , Expressão Gênica , Humanos , Hidroxilação , Saccharomyces cerevisiae/metabolismo
16.
Bioorg Med Chem Lett ; 27(16): 3683-3687, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28711350

RESUMO

Inhibitors of CYP1 enzymes may play vital roles in the prevention of cancer and overcoming chemo-resistance to anticancer drugs. In this letter, we report synthesis of twenty-three pyrrole based heterocyclic chalcones which were screened for inhibition of CYP1 isoforms. Compound 3n potently inhibited CYP1B1 with an IC50 of ∼0.2µM in Sacchrosomes™ and CYP1B1-expressing live human cells. However, compound 3j which inhibited both CYP1A1 and CYP1B1 with an IC50 of ∼0.9µM, using the same systems, also potently antagonized B[a]P-mediated induction of AhR signaling in yeast (IC50, 1.5µM), fully protected human cells from B[a]P toxicity and completely reversed cisplatin resistance in human cells that overexpress CYP1B1 by restoring cisplatin's cytotoxicity. Molecular modeling studies were performed to rationalize the observed potency and selectivity of enzyme inhibition by compounds 3j and 3n.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Chalconas/síntese química , Chalconas/farmacologia , Citocromo P-450 CYP1B1/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Antineoplásicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP1B1/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Pirróis/síntese química , Pirróis/farmacologia , Relação Estrutura-Atividade
17.
J Chem Inf Model ; 57(6): 1309-1320, 2017 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-28489395

RESUMO

Target structure-guided virtual screening (VS) is a versatile, powerful, and inexpensive alternative to experimental high-throughput screening (HTS). To discover potent CYP1A1 enzyme inhibitors for cancer chemoprevention, a commercial library of 50 000 small molecules was utilized for VS guided by both ligand and structure-based strategies. For experimental validation, 300 ligands were proposed based on combined analysis of fitness scores from ligand based e-pharmacophore screening and docking score, prime MMGB/SA binding affinity and interaction pattern analysis from structure-based VS. These 300 compounds were screened, at 10 µM concentration, for in vitro inhibition of CYP1A1-Sacchrosomes (yeast-derived microsomal enzyme) in the ethoxyresorufin-O-de-ethylase assay. Thirty-two compounds displayed >50% inhibition of CYP1A1 enzyme activity at 10 µM. 2-Phenylimidazo-[1,2-a]quinoline (5121780, 119) was found to be the most potent with 97% inhibition. It also inhibited ∼95% activity of CYP1B1 and CYP1A2, the other two CYP1 enzymes. The compound 5121780 (119) showed high selectivity toward inhibition of CYP1 enzymes with respect to CYP2 and CYP3 enzymes (i.e., there was no detectable inhibition of CYP2D6/CYP2C9/CYP2C19 and CYP3A4 at 10 µM). It was further investigated in live CYP-expressing human cell system, which confirmed that compound 5121780 (119) potently inhibited CYP1A1, CYP1A2, CYP1B1 enzymes with IC50 values of 269, 30, and 56 nM, respectively. Like in Sacchrosomes, inhibition of CYP2D6/CYP2C9/CYP2C19 and CYP3A4 enzymes, expressed within live human cells, could hardly be detected at 10 µM. The compound 119 rescued CYP1A1 overexpressing HEK293 cells from CYP1A1 mediated benzo[a]pyrene (B[a]P) toxicity and also overcame cisplatin resistance in CYP1B1 overexpressing HEK293 cells. Molecular dynamics simulations of 5121780 (119) with CYP1 enzymes was performed to understand the interaction pattern to CYP isoforms. Results indicate that VS can successfully be used to identify promising CYP1A1 inhibitors, which may have potential in the development of novel cancer chemo-preventive agents.


Assuntos
Citocromo P-450 CYP1A1/antagonistas & inibidores , Inibidores das Enzimas do Citocromo P-450/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Microssomos/efeitos dos fármacos , Interface Usuário-Computador , Leveduras/genética , Sobrevivência Celular , Citocromo P-450 CYP1A1/química , Citocromo P-450 CYP1A1/metabolismo , Células HEK293 , Humanos , Ligantes , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica
18.
Eur J Med Chem ; 130: 320-327, 2017 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-28259840

RESUMO

CYP1B1 is implicated to have a role in the development of breast, ovarian, renal, skin and lung carcinomas. It has been suggested that identification of potent and specific CYP1B1 inhibitors can lead to a novel treatment of cancer. Flavonoids have a compact rigid skeleton which fit precisely within the binding cavity of CYP1B1. Systematic isosteric replacement of flavonoid 'O' atom with 'N' atom led to the prediction that a 'quinazoline' scaffold could be the basis for designing potential CYP1B1 inhibitors. A total of 20 quinazoline analogs were synthesized and screened for CYP1B1 and CYP1A1 inhibition in Sacchrosomes™. IC50 determinations of six compounds with capability of inhibiting CYP1B1 identified quinazolines 5c and 5h as the best candidates for CYP1B1 inhibition, with IC50 values in the nM range. Further selectivity studies with homologous CYPs, belonging to the CYP1, CYP2 and CYP3 family of enzymes, showed that the compounds are likely to be free from critical drug-drug interaction liability. Molecular modelling studies were performed to rationalize the observed enzymatic inhibitions. Further biological studies in live yeast and human cells, harboring CYP1A1 and CYP1B1 enzymes, have illustrated the most potent compounds' cellular permeability and capability of potently inhibiting CYP1B1 enzyme expressed within live cells.


Assuntos
Antineoplásicos/química , Citocromo P-450 CYP1B1/antagonistas & inibidores , Quinazolinas/farmacologia , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Permeabilidade da Membrana Celular , Células Cultivadas , Citocromo P-450 CYP1A1 , Família 2 do Citocromo P450/efeitos dos fármacos , Família 3 do Citocromo P450/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Modelos Moleculares , Quinazolinas/química , Quinazolinas/farmacocinética , Leveduras/citologia
19.
Eur J Med Chem ; 129: 159-174, 2017 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-28222316

RESUMO

The structure of alpha-napthoflavone (ANF), a potent inhibitor of CYP1A1 and CYP1B1, mimics the structure of chalcones. Two potent CYP1B1 inhibitors 7k (DMU2105) and 6j (DMU2139) have been identified from two series of synthetic pyridylchalcones. They inhibit human CYP1B1 enzyme bound to yeast-derived microsomes (Sacchrosomes™) with IC50 values of 10 and 9 nM, respectively, and show a very high level of selectivity towards CYP1B1 with respect to the IC50 values obtained with CYP1A1, CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2C19 Sacchrosomes™. Both compounds also potently inhibit CYP1B1 expressed within 'live' recombinant yeast and human HEK293 kidney cells with IC50 values of 63, 65, and 4, 4 nM, respectively. Furthermore, the synthesized pyridylchalcones possess better solubility and lipophilicity values than ANF. Both compounds overcome cisplatin-resistance in HEK293 and A2780 cells which results from CYP1B1 overexpression. These potent cell-permeable and water-soluble CYP1B1 inhibitors are likely to have useful roles in the treatment of cancer, glaucoma, ischemia and obesity.


Assuntos
Chalconas/farmacologia , Inibidores Enzimáticos/farmacocinética , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Chalconas/síntese química , Chalconas/farmacocinética , Cisplatino , Citocromo P-450 CYP1B1/antagonistas & inibidores , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Células HEK293 , Compostos Heterocíclicos , Humanos
20.
Org Biomol Chem ; 14(38): 8931-8936, 2016 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-27714268

RESUMO

Highly selective CYP1B1 inhibitors have potential in the treatment of hormone-induced breast and prostate cancers. Mimicry of potent and selective CYP1B1 inhibitors, α-naphthoflavone and stilbenes, revealed that two sets of hydrophobic clusters suitably linked via a polar linker could be implanted into a new scaffold 'biphenyl ureas' to create potentially a new class of CYP1B1 inhibitors. A series of sixteen biphenyl ureas were synthesized and screened for CYP1B1 and CYP1A1 inhibition in Sacchrosomes™, yeast-derived recombinant microsomal enzymes. The most active human CYP1B1 inhibitors were further studied for their selectivity against human CYP1A1, CYP1A2, CYP3A4 and CYP2D6 enzymes. The meta-chloro-substituted biphenyl urea 5h was the most potent inhibitor of CYP1B1 with IC50 value of 5 nM. It displayed excellent selectivity over CYP1A1, CYP1A2, CYP3A4 and CYP2D6 (IC50 >10 µM in the four CYP assays, indicating >2000-fold selectivity). Similarly, two methoxy-substituted biphenyl ureas 5d and 5e also displayed potent and selective inhibition of CYP1B1 with IC50 values of 69 and 58 nM, respectively, showing >62 and >98-fold selectivity over CYP1A1, CYP1A2, CYP3A4 and CYP2D6 enzymes. In order to probe if the relatively insoluble biphenyl ureas were cell permeable and if they could at all be used for future cellular studies, their CYP1B1 inhibition was investigated in live recombinant human and yeast cells. Compound 5d displayed the most potent inhibition with IC50s of 20 nM and 235 nM, respectively, in the two cell-based assays. The most potent and selective CYP1B1 inhibitor (compound 5h) from Sacchrosomes, also displayed potent inhibition in live cell assays. Molecular modeling was performed to understand the trends in potency and selectivity observed in the panel of five CYP isoenzymes used for the in vitro studies.


Assuntos
Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Citocromo P-450 CYP1B1/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/farmacologia , Citocromo P-450 CYP1B1/metabolismo , Células HEK293 , Halogenação , Humanos , Modelos Moleculares , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo
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