The Effect of Dual Bioactive Compounds Artemisinin and Metformin Co-loaded in PLGA-PEG Nano-particles on Breast Cancer Cell lines: Potential Apoptotic and Anti-proliferative Action.

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Perspective of structural flexibility on selective inhibition towards CYP1B1 over CYP1A1 by α-naphthoflavone analogs.

Research on action selectivity between CYP1A1 and CYP1B1 is particularly valuable for cancer chemoprevention and chemotherapy. However, they share a very close similarity in their ligand-binding pockets that α-naphthoflavone (ANF) is the co-crystal ligand for both isoforms, which poses a major challenge in revealing their selectivity mechanism. Therefore, three selective CYP1B1 inhibitors derived from ANF were selected to illustrate the structural basis for the selectivity between the two isoforms via a comprehensive computational strategy. It was found that the sustainability of the π-π stacking interactions with the phenylalanine residues of the two isoforms, namely, Phe123, Phe224, and Phe258 for CYP1A1, and Phe134, Phe231, and Phe268 for CYP1B1, played a crucial role in determining the selectivity of ligands with a classic aromatic conjugation system like ANF and its derivatives for CYP1B1 versus CYP1A1. Of note, the structural flexibility of the corresponding protein domains mainly orchestrated the sustainability of the corresponding π-π stacking interactions, thereby determining the binding selectivity. Therefore, the structure modification of naphthoflavone lead compounds into preferable binding configurations to satisfy the π-π stacking interactions of the key phenylalanine residues within CYP1B1 would be an inspiring strategy devised to improve the inhibitory selectivity towards CYP1B1. Collectively, this study revealed valuable insight into understanding the selective mechanism between CYP1A1 and CYP1B1 from the perspective of structural flexibility, which sheds light on the future rational design of CYP1B1 selective inhibitors.

Exposure of Physalaemus cuvieri (Anura) to benzo[a]pyrene and α-naphthoflavone: Morphofunctional effects on hepatic melanomacrophages and erythrocytes abnormalities.

Benzo[a]pyrene (BaP) is a high-risk contaminant of elevated toxicity. Its biotransformation process occurs as the expression of CYP1A1 increases and produces toxic metabolites. In turn, α-naphthoflavone (aNF) represents an inhibitor of CYP1A1, preventing BaP metabolism. Toxicological studies in anurans show alterations in the melanomacrophage (MM) detoxification cell after exposure to xenobiotics. In this study, the production of melanin by MMs was evaluated, as were morphological alterations in the cytoskeleton, phagocytosis and the genotoxicity effects after exposure of an anuran species to BaP and aNF. Physalaemus cuvieri received subcutaneous injections of 2 mg/kg and/or 20 mg/kg aNF. For phagocytosis analyses, animals received an intraperitoneal injection with 0.4% trypan blue. The results revealed that melanin synthesis increased by 503.2% in animals exposed to BaP after 48 h, which was related to the antioxidant action of melanin, whereas the decreased in synthesis of 25.6% with the BaP + aNF interaction resulted in high toxicity to MMs and cell degeneration. The phagocytic activity reduced to 37.6% in animals exposed to BaP, characterizing a functional impairment; however, the BaP + aNF interaction led to the restoration of phagocytosis, reaching 419.23%. The decreased rate or absence of abnormalities may be explained by the fact that only the less damaged erythrocytes remained in the bloodstream, whereas the most damaged cells died. In conclusion, BaP and aNF are toxic to P. cuvieri, bringing risks to herpetofauna.

α-naphthoflavone-derived cytochrome P450 (CYP)1B1 degraders specific for sensitizing CYP1B1-mediated drug resistance to prostate cancer DU145: Structure activity relationship.

We previously discovered extrahepatic cytochrome P450 1B1 (CYP1B1) degraders able to overcome drug resistance toward docetaxel using a PROTACs technology, however, the underexplored structure activity relationships and poor water solubility posed a major hurdle in the development of CYP1B1 degraders. Herein, continuous efforts are made to develop more promising α-naphthoflavone (ANF)-derived chimeras for degrading CYP1B1. Guided by the strongest ANF-derived CYP1B1 degrader 3a we ever reported, 17 ANF analogues are designed and synthesized to evaluate the CYP1B1 degradation and resultant resistance reversal. In degrading CYP1B1 and sensitizing drug resistance, 4d with a 1, 5-cis triazole coupling mode at (C3') of B ring of ANF exhibited the similar potency as 3a carrying a 1, 4-trans triazole fragment at (C4') of B ring, but more obvious selectivity of 4d toward CYP1B1 over CYP1A2 is observed. When an oxygen was inserted into the linker of 4d, 4f demonstrated better water solubility, a more potent ability in degrading CYP1B1 and reversing drug resistance, and a promising selectivity. Collectively, a substitution position, an alkyne-azide cyclization and a liker type significantly affect the ability of ANF-thalidomide conjugates in eliminating drug resistance of CYP1B1-expressing DU145 (DU145/CY) cells to docetaxel via targeted CYP1B1 degradation.

Comparison of the Stimulatory and Inhibitory Effects of Steroid Hormones and α-Naphthoflavone on Steroid Hormone Hydroxylation Catalyzed by Human Cytochrome P450 3A Subfamilies.

The inhibitory and stimulatory effects of steroid hormones and related compounds on the hydroxylation activity at the 6ß-position of two steroid hormones, progesterone and testosterone, by CYP3A4, polymorphically expressed CYP3A5, and fetal CYP3A7 were compared to clarify the catalytic properties of the predominant forms of the human CYP3A subfamily. Hydroxylation activities of progesterone and testosterone by CYP3A4, CYP3A5, and CYP3A7 were estimated using HPLC. The Michaelis constants (Km) for progesterone 6ß-hydroxylation by CYP3A5 were markedly decreased in the presence of dehydroepiandrosterone (DHEA) and α-naphthoflavone (ANF), whereas progesterone and DHEA competitively inhibited testosterone 6ß-hydroxylation mediated by CYP3A4, and progesterone competitively inhibited CYP3A5-mediated activity, which was weaker than that for CYP3A4. ANF noncompetitively inhibited testosterone 6ß-hydroxylation mediated by both CYP3A4 and CYP3A5. Progesterone and testosterone 6ß-hydroxylation mediated by CYP3A7 was inhibited or unaffected by DHEA, pregnenolone, and ANF. These results suggested that DHEA and ANF stimulated progesterone 6ß-hydroxylation by CYP3A5 but not by CYP3A4 and CYP3A7; however, progesterone, DHEA, and ANF inhibited testosterone 6ß-hydroxylation mediated by all CYP3A subfamily members. The inhibitory/stimulatory pattern of steroid-steroid interactions is different among CYP3A subfamily members and CYP3A5 is the most sensitive in terms of activation among the CYP3A subfamily members investigated.

Discovery of heterocycle-containing α-naphthoflavone derivatives as water-soluble, highly potent and selective CYP1B1 inhibitors.

Cytochrome P450 1B1 (CYP1B1) has been well validated as an attractive target for cancer prevention and drug resistance reversal. In continuation of our interest in this area, herein, a set of forty-six 6,7,10-trimethoxy-α-naphthoflavone derivatives varying in B ring was synthesized and screened against CYP1 enzymes, leading to the identification of fluorine-containing compound 15i as the most potent and selective CYP1B1 inhibitor (IC50 value of 0.07 nM), being 84-fold more potent than that of the template molecule ANF. Alternatively, the amino-substituted derivative 13h not only possessed a potent inhibitory effect on CYP1B1 (IC50 value of 0.98 nM), but also had a substantially increased water solubility as compared with the lead ANF (311 µg/mL for 13h and <5 µg/mL for ANF). The current study expanded the structural diversity of CYP1B1 inhibitors, and compound 13h could be considered as a promising starting point with great potential for further studies.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4.

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

Structure of an ancestral mammalian family 1B1 cytochrome P450 with increased thermostability.

Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable, with T50 values of ∼30-40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared with related extant forms, but they also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. Whereas extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17ß-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability. To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to its thermostability.

Inhibition of human CYP1 enzymes by a classical inhibitor α-naphthoflavone and a novel inhibitor N-(3, 5-dichlorophenyl)cyclopropanecarboxamide: An in vitro and in silico study.

Enzymes in the cytochrome P450 family 1 (CYP1) catalyze metabolic activation of procarcinogens and deactivation of certain anticancer drugs. Inhibition of these enzymes is a potential approach for cancer chemoprevention and treatment of CYP1-mediated drug resistance. We characterized inhibition of human CYP1A1, CYP1A2, and CYP1B1 enzymes by the novel inhibitor N-(3,5-dichlorophenyl)cyclopropanecarboxamide (DCPCC) and α-naphthoflavone (ANF). Depending on substrate, IC50 values of DCPCC for CYP1A1 or CYP1B1 were 10-95 times higher than for CYP1A2. IC50 of DCPCC for CYP1A2 was 100-fold lower than for enzymes in CYP2 and CYP3 families. DCPCC IC50 values were 10-680 times higher than the ones of ANF. DCPCC was a mixed-type inhibitor of CYP1A2. ANF was a competitive tight-binding inhibitor of CYP1A1, CYP1A2, and CYP1B1. CYP1A1 oxidized DCPCC more rapidly than CYP1A2 or CYP1B1 to the same metabolite. Molecular dynamics simulations and binding free energy calculations explained the differences of binding of DCPCC and ANF to the active sites of all three CYP1 enzymes. We conclude that DCPCC is a more selective inhibitor for CYP1A2 than ANF. DCPCC is a candidate structure to modulate CYP1A2-mediated metabolism of procarcinogens and anticancer drugs.

Design and synthesis of α-naphthoflavone chimera derivatives able to eliminate cytochrome P450 (CYP)1B1-mediated drug resistance via targeted CYP1B1 degradation.

Extrahepatic cytochrome P450 1B1 (CYP1B1), which is highly expressed in various tumors, is an attractive and potential target for cancer prevention, therapy, and reversal of drug resistance. CYP1B1 inhibition is the current predominant therapeutic paradigm to treating CYP1B1-mediated malignancy, but therapeutic effect has little success. Herein, we reported CYP1B1 degradation in place of CYP1B1 inhibition for reversing drug resistance toward docetaxel in CYP1B1-overexpressing prostate cancer cell line DU145 using a PROTAC strategy. Replacing chlorine atom of a CYP1B1 selective inhibitor we found previously with ethynyl, we got the resulting α-naphthoflavone derivative 5 which kept strong inhibition against CYP1B1 (IC50 = 0.4 ± 0.2 nM) and high selectivity. Coupling of 5 with thalidomide derivatives of varying chain lengths afforded conjugates 6A-Dvia click reaction. In vitro cell-based assay indicated that 6C was more effective in eliminating drug resistance of CYP1B1-overexpressed DU145 cells compared with other analogues. Western blotting analysis showed CYP1B1 degradation was one main reason for the reversal of drug resistance to docetaxel and the effect was obtained in a concentration-dependent manner. This work is the first attempt to overcome CYP1B1-mediated drug resistance via CYP1B1 degradation instead of CYP1B1 inhibition, which could provide a new direction toward eliminating drug resistance.

Preventative effects of alpha-naphtho flavone in vascular dementia.

Vascular dementia (VaD) refers to the loss of reasoning, planning, judgment, and memory as a result of reduced blood flow, inflammation, activation of immune cells, and neurodegenerative processes. Here, we tested the preventive effects of alpha-naphthoflavone (ANF) on a model of vascular dementia induced by four-vessel occlusion (4VO) in rats. Animals were treated orally for 5 consecutive days with 10 mg/kg of donepezil (a traditional therapeutic drug) and 40 and 80 mg/kg ANF. The treatment with ANF exerted effects that were similar to those induced by donepezil. This included prevention of cognitive impairment, alterations in the plasma levels of homocysteine and nitrate and the activity of acetylcholinesterase and myeloperoxidase and the tissue level of glutathione, and thiobarbituric acid reactive substances in the brain tissues. These findings suggest that herbal-derived ANF is as effective as traditional drugs in vascular dementia.

3-Methylcholanthrene impacts on the female germ cells of rats without causing systemic toxicity.

We have previously shown that daily exposure to the environmental pollutant 3-methylcholanthrene (3MC) alters the ovarian function by affecting follicle growth and ovulation. To extend our findings, the aims of this work were to study the effects of daily and non-daily exposure to 3MC on oocyte morphology and integrity and the meiosis process. To this end, immature female rats were daily (0.1-1.0 mg/kg) and non-daily (0.1 mg/kg, three times a week) exposed to 3MC and/or α-naphthoflavone (αNF) (80 mg/kg) for 19 and 20 days, respectively. The latter was used to study its ability to prevent the 3MC action. Follicular growth was examined by histology, apoptosis by in situ cell death detection, oocyte integrity by morphological parameters and fluorescent dyes, and the meiotic spindle by immunostaining. Compared with controls (C), and in a dose-dependent manner, all 3MC-treated rats showed i) increased presence of apoptotic cells in antral follicles and decreased percentage of healthy oocytes, ii) increased oocyte area, perimeter and perivitelline space and decreased thickness of the zona pellucida, and ii) increased percentage of oocytes with abnormal meiotic spindle. In addition, the non-daily dose of 3MC caused DNA damage in oocytes, but not in blood or bone marrow cells. All 3MC-induced changes were prevented with the co-treatment with αNF. These results suggest that low doses of 3MC severely disrupt the ovarian function and that germ cells seem to be more sensitive to this environmental pollutant than other cells such as peripheral blood and bone marrow cells.

Synthesis and structure-activity relationship studies of α-naphthoflavone derivatives as CYP1B1 inhibitors.

Cytochrome P450 1B1(CYP1B1) has been recognized as an important target for cancer prevention and drug resistance reversal. In order to obtain potent and selective CYP1B1 inhibitors, a series of forty-one α-naphthoflavone (ANF) derivatives were synthesized, characterized, and evaluated for CYP1B1, CYP1A1 and CYP1A2 inhibitory activities. A closure look into the structure-activity relationship for the inhibitory effects on CYP1B1 indicated that modification of the C ring of ANF would decrease the CYP1B1 inhibitory potency, while incorporation of substituent(s) into the different positions of the B ring yielded analogues with varying CYP1B1 inhibitory capacity. Among these derivatives, compounds 9e and 9j were identified as the most potent two selective CYP1B1 inhibitors with IC50 values of 0.49 and 0.52 nM, respectively, which were 10-fold more potent than the lead compound ANF. In addition, molecular docking and a reasonable 3D-QSAR (three-dimensional quantitative structure-activity relationship) study were performed to provide a better understanding of the key structural features influencing the CYP1B1 inhibitory activity. The results achieved in this study would lay a foundation for future development of selective, potent, low-toxic and water-soluble CYP1B1 inhibitors.

Alpha-naphthoflavone attenuates non-alcoholic fatty liver disease in oleic acid-treated HepG2 hepatocytes and in high fat diet-fed mice.

Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease. The literature suggests that the aryl hydrocarbon receptor (AHR) may be a key player in the pathogenesis of NAFLD, and it can modulate the synthesis of cytochrome P450 1A1 (CYP1A1) and tumor necrosis factor-α (TNF-α). Previous studies have shown that CYP1A1 is a key enzyme of oxidative stress, TNF-α is involved in the formation of insulin resistance (IR), oxidative stress and insulin resistance are the key factors for the formation of NAFLD. Therefore, it can be said that AHR may participate in contributing to NAFLD by regulating CYP1A1 and TNF-α. Alpha-naphthoflavone (ANF) is an effective AHR inhibitor. The present study was designed to explore the hepatoprotective effect of ANF in high fat diet (HFD)-induced NAFLD mice and oleic acid (OA)-treated HepG2 hepatocytes. Mice were fed HFD to induce NAFLD, HepG2 cells were exposed to OA to induce hepatocyte injury, and ANF significantly reduced mouse and cellular liver damage compared to the HFD-induced NAFLD and OA-treated HepG2 hepatocytes. ANF treatment reduces liver damage by reducing ROS and IR, the data show that ANF inhibits the expression of AHR, CYP1A1 and TNF-α in NAFLD. Taken together, these findings show that ANF alleviate NAFLD via regulation of AHR/CYP1A1 and AHR/TNF-α pathways, which may have potential for further development as novel therapeutic agents for NAFLD.

The Aryl hydrocarbon receptor mediates tobacco-induced PD-L1 expression and is associated with response to immunotherapy.

Whether tobacco carcinogens enable exposed cells immune escape resulting in carcinogenesis, and why patients who smoke respond better to immunotherapies than non-smokers, remains poorly understood. Here we report that cigarette smoke and the carcinogen benzo(a)pyrene (BaP) induce PD-L1 expression on lung epithelial cells in vitro and in vivo, which is mediated by aryl hydrocarbon receptor (AhR). Anti-PD-L1 antibody or deficiency in AhR significantly suppresses BaP-induced lung cancer. In 37 patients treated with anti-PD-1 antibody pembrolizumab, 13/16 (81.3%) patients who achieve partial response or stable disease express high levels of AhR, whereas 12/16 (75%) patients with progression disease exhibit low levels of AhR in tumor tissues. AhR inhibitors exert significant antitumor activity and synergize with anti-PD-L1 antibody in lung cancer mouse models. These results demonstrate that tobacco smoke enables lung epithelial cells to escape from adaptive immunity to promote tumorigenesis, and AhR predicts the response to immunotherapy and represents an attractive therapeutic target.

Alpha-naphthoflavone induces apoptosis through endoplasmic reticulum stress via c-Src-, ROS-, MAPKs-, and arylhydrocarbon receptor-dependent pathways in HT22 hippocampal neuronal cells.

α-Naphthoflavone (αNF) is a prototype flavone, also known as a modulator of aryl hydrocarbon receptor (AhR). In the present study, we investigated the molecular mechanisms of αNF-induced cytotoxic effects in HT22 mouse hippocampal neuronal cells. αNF induced apoptotic cell death via activation of caspase-12 and -3 and increased expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by treatment with the ER stress inhibitor, salubrinal, or by CHOP siRNA transfection reduced αNF-induced cell death. αNF activated mitogen-activated protein kinases (MAPKs), such as p38, JNK, and ERK, and inhibition of MAPKs reduced αNF-induced CHOP expression and cell death. αNF also induced accumulation of reactive oxygen species (ROS) and an antioxidant, N-acetylcysteine, reduced αNF-induced MAPK phosphorylation, CHOP expression, and cell death. Furthermore, αNF activated c-Src kinase, and inhibition of c-Src by a kinase inhibitor, SU6656, or siRNA transfection reduced αNF-induced ROS accumulation, MAPK activation, CHOP expression, and cell death. Inhibition of AhR by an AhR antagonist, CH223191, and siRNA transfection of AhR and AhR nuclear translocator reduced αNF-induced AhR-responsive luciferase activity, CHOP expression, and cell death. Finally, we found that inhibition of c-Src and MAPKs reduced αNF-induced transcriptional activity of AhR. Taken together, these findings suggest that αNF induces apoptosis through ER stress via c-Src-, ROS-, MAPKs-, and AhR-dependent pathways in HT22 cells.

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes.

Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3-phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3-methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a non-selective fashion. The advantages of the novel substrates were (1) an excellent signal-to-background ratio, (2) selectivity for CYP1 forms, and (3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

Design and synthesis of selective CYP1B1 inhibitor via dearomatization of α-naphthoflavone.

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.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces alterations in myogenic differentiation of C2C12 cells.

Dioxin-induced toxicities that affect the development of the motor system have been proposed since many years. However, cellular evidence and the molecular basis for the effects are limited. In this study, a cultured mouse myoblast cell line, C2C12, was utilized to examine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on myogenic differentiation and expression of acetylcholinesterase (AChE), a neuromuscular transmission-related gene. The results showed that TCDD exposure at 10-10 M repressed the myotube formation of C2C12 cells by disturbing the fusion process and suppressing the expression of myosin heavy chain, a myobute structural protein, and not by induction of cytotoxicity. Furthermore, TCDD dose dependently suppressed the transcriptional expression and enzymatic activity of AChE during the myogenic differentiation, particularly in the middle stage. However, the administration of aryl hydrocarbon receptor antagonists, CH223191 and alpha-naphthoflavone, did not completely reverse the TCDD-induced downregulation of muscular AChE during myogenic differentiation. These findings suggest that low dose exposure to dioxin may result in disturbances of muscle differentiation and neuromuscular transmission.

Application of α- and ß-naphthoflavones as monooxygenase inhibitors of Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651 in transformation of 17α-methyltestosterone.

In this work, 17α-methyltestosterone was effectively hydroxylated by Absidia coerulea KCh 93, Syncephalastrum racemosum KCh 105 and Chaetomium sp. KCh 6651. A. coerulea KCh 93 afforded 6ß-, 12ß-, 7α-, 11α-, 15α-hydroxy derivatives with 44%, 29%, 6%, 5% and 9% yields, respectively. S. racemosum KCh 105 afforded 7α-, 15α- and 11α-hydroxy derivatives with yields of 45%, 19% and 17%, respectively. Chaetomium sp. KCh 6651 afforded 15α-, 11α-, 7α-, 6ß-, 9α-, 14α-hydroxy and 6ß,14α-dihydroxy derivatives with yields of 31%, 20%, 16%, 7%, 5%, 7% and 4%, respectively. 14α-Hydroxy and 6ß,14α-dihydroxy derivatives were determined as new compounds. Effect of various sources of nitrogen and carbon in the media on biotransformations were tested, however did not affect the degree of substrate conversion or the composition of the products formed. The addition of α- or ß-naphthoflavones inhibited 17α-methyltestosterone hydroxylation but did not change the percentage composition of the resulting products.