Biotransformation, Using Recombinant CYP450-Expressing Baker's Yeast Cells, Identifies a Novel CYP2D6.10A122V Variant Which Is a Superior Metabolizer of Codeine to Morphine Than the Wild-Type Enzyme

© Copyright 2018 American Chemical Society. 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

Khellinoflavanone, a Semisynthetic Derivative of Khellin, Overcomes Benzo[ a]pyrene Toxicity in Human Normal and Cancer Cells That Express CYP1A1

Copyright © 2018 American Chemical Society. 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.

Nonantioxidant Tetramethoxystilbene Abrogates α-Synuclein-Induced Yeast Cell Death but Not That Triggered by the Bax or βa4 Peptide

© 2018 American Chemical Society. 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 βA4, 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

Biotransformation of Chrysin to Baicalein: Selective C6-Hydroxylation of 5,7-Dihydroxyflavone Using Whole Yeast Cells Stably Expressing Human CYP1A1 Enzyme

© 2017 American Chemical Society. 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

Furanoflavones pongapin and lanceolatin B blocks the cell cycle and induce senescence in CYP1A1-overexpressing breast cancer cells

© 2018 Elsevier Ltd 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

Glycyrrhiza glabra extract and quercetin reverses cisplatin resistance in triple-negative MDA-MB-468 breast cancer cells via inhibition of cytochrome P450 1B1 enzyme

© 2017 Elsevier Ltd 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

Synthesis and biological evaluation of pyrrole-based chalcones as CYP1 enzyme inhibitors, for possible prevention of cancer and overcoming cisplatin resistance

© 2017 Elsevier Ltd 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

Biphenyl urea derivatives as selective CYP1B1 inhibitors

© 2016 The Royal Society of Chemistry. 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

Discovery and characterization of novel CYP1B1 inhibitors based on heterocyclic chalcones: Overcoming cisplatin resistance in CYP1B1-overexpressing lines

© 2017 Elsevier Masson SAS 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 IC 50 values of 10 and 9nM, respectively, and show a very high level of selectivity towards CYP1B1 with respect to the IC 50 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 IC 50 values of 63, 65, and 4, 4nM, respectively. Furthermore, the synthesized pyridylchalcones possess better solubility and lipophilicity values than ANF. Both compounds overcome cisplatin–resistance in HEK293 and A2780cells 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

(E)-3-(3,4,5-Trimethoxyphenyl)-1-(pyridin-4-yl)prop-2-en-1-one, a heterocyclic chalcone is a potent and selective CYP1A1 inhibitor and cancer chemopreventive agent

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