Cytochrome P450 1B1 Inhibition Research Articles

Development of novel N-aryl-2,4-bithiazole-2-amine-based CYP1B1 degraders for reversing drug resistance

Extrahepatic cytochrome P450 1B1 (CYP1B1), which is highly expressed in non-small cell lung cancer, is an attractive target for cancer prevention, therapy, and overcoming drug resistance. Historically, CYP1B1 inhibition has been the primary therapeutic approach for treating CYP1B1-related malignancies, but its success has been limited. This study introduced CYP1B1 degradation as an alternative strategy to counter drug resistance and metastasis in CYP1B1-overexpressing non-small cell lung cancer A549/Taxol cells via a PROTAC strategy. Our investigation revealed that the identification of the potent CYP1B1 degrader PV2, achieving DC50 values of 1.0 nM and inducing >90 % CYP1B1 degradation at concentrations as low as 10 nM in A549/Taxol cells. Importantly, PV2 enhanced the sensitivity of the A549/Taxol subline to Taxol, possibly due to its stronger inhibitory effects on P-gp through CYP1B1 degradation. Additionally, compared to the CYP1B1 inhibitor A1, PV2 effectively suppressed the migration and invasion of A549/Taxol cells by inhibiting the FAK/SRC and EMT pathways. These findings hold promise for a novel therapy targeting advanced CYP1B1+ non-small cell lung cancer.

A new class of CYP1B1 inhibitors derived from bentranil

Cytochrome P450 1B1 (CYP1B1) is highly expressed in a variety of tumors and implicated to drug resistance. More and more researches have suggested that CYP1B1 is a new target for cancer prevention and therapy. Various CYP1B1 inhibitors with a rigid polycyclic skeleton have been developed, such as flavonoids, trans-stilbenes, and quinazolines. To obtain a new class of CYP1B1 inhibitors, we designed and synthesized a series of bentranil analogues, moreover, IC50 determinations were performed for CYP1B1 inhibition of five of these compounds and found that 6o and 6q were the best inhibitors, with IC50 values in the nM range. The selectivity index (SI) of CYP1B1 over CYP1A1 and CYP1A2 was 30-fold higher than that of α-naphthoflavone (ANF). The molecular docking results showed that compound 6q fitted better into the CYP1B1 binding site than other compounds, which was consistent with our experimental results. On the basis of 6o and 6q, it is expected to develop CYP1B1 inhibitors with stronger affinity, higher selectivity and better solubility.

Design of Cytochrome P450 1B1 Inhibitors via a Scaffold-Hopping Approach.

Cytochrome P450 1B1 (CYP1B1) is a potential drug target in cancer research that is overexpressed in several solid tumors but is present only at low levels in healthy tissues. Its expression is associated with resistance to common chemotherapeutics, while inhibitors restore efficacy to these drugs in model systems. The majority of CYP1B1 inhibitors are derived from a limited number of scaffolds, and few have achieved outstanding selectivity against other human CYPs, which could impede clinical development. This study explores a new chemical space for CYP1B1 inhibitors using a scaffold-hopping approach and establishes 2,4-diarylthiazoles as a promising framework for further development. From a small library, compound 15 emerged as the lead, with picomolar CYP1B1 inhibition, and over 19,000-fold selectivity against its relative, CYP1A1. To investigate the activity of 15, molecular dynamics, optical spectroscopy, point mutations, and traditional structure-activity relationships were employed and revealed key interactions important for the development of CYP1B1 inhibitors.

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.

Fluconazole Inhibits Cytochrome P450 1B1 and its Associated Arachidonic Acid Metabolites in the Heart and Protects against Angiotensin II‐induced Cardiac Hypertrophy

Cytochrome P450 1B1 (CYP1B1) has been reported to have a major role in metabolizing arachidonic acid (AA) into cardiotoxic metabolites, mid‐chain hydroxyeicosatetraenoic acid (HETEs). Of particular interest, several studies have demonstrated the role of mid‐chain HETEs in the development of cardiac hypertrophy. Recently, we have shown that fluconazole inhibits the formation of mid‐chain HETEs metabolite, however, whether fluconazole would be able to modulate CYP1B1‐mediated AA metabolism in an Ang II‐induced cellular hypertrophy model and in rats' heart has never been investigated before. Therefore, the objectives of this study were; 1) to investigate the effect of fluconazole on CYP1B1 mediated AA metabolites and to explore the potential protective effect against Ang II‐induced cellular hypertrophy, and 2) to study the effect of fluconazole on CYP1B1 and its associated AA metabolites in vivo in Sprague Dawley rats. For this purpose, both H9c2 and RL‐14 cells were treated with 10 μM Ang II in the absence and presence of 50 μM fluconazole for 24 hours. Also, Sprague Dawley rats (n=6) were injected intraperitoneally with a single dose of fluconazole (20 mg/kg) or saline and the heart tissues were harvested 24 hours post‐treatment. Thereafter, the formation of AA metabolites was measured using liquid chromatography‐electron spray ionization‐mass spectrometry (LC‐ESI‐MS). Whereas, the expression of cardiac hypertrophic markers and CYP1B1 were determined by real time‐polymerase chain reaction and Western blot analysis, respectively. Moreover, enzymatic activity and determination of CYP1B1 inhibition kinetics by fluconazole were assessed using 7‐ethoxyresorufin O‐deethylase (EROD) and recombinant human CYP1B1 assays. Our results demonstrated that fluconazole was able to attenuate Ang‐II‐induced cellular hypertrophy as evidenced by a significant inhibition of hypertrophic markers, β‐myosin heavy chain (MHC)/ α‐MHC, BNP as well as cell surface area. The protective effect of fluconazole was associated with a significant decrease in the level of CYP1B1 gene, protein, activity levels and its associated mid‐chain HETEs metabolite induced by Ang II. Furthermore, treatment of rats with fluconazole significantly decreased the expression of CYP1B1 enzyme and the formation level of cardiotoxic mid‐chain HETEs metabolites in the heart. In conclusion, our results showed that fluconazole protects against Ang II‐induced cellular hypertrophy by inhibiting CYP1B1 and its associated mid‐chain HETEs metabolites. Also, fluconazole may be repurposed as a mid‐chain HETEs formation inhibitor for the treatment of cardiac hypertrophy and heart failure.Support or Funding InformationThis work was supported by a grant from the Canadian Institutes of Health Research [Grant 106665] to A.O.S.E. A.H.A is the recipient of Saudi Arabian government scholarship provided by Qassim University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of 19-(S/R)Hydroxyeicosatetraenoic Acid as the First Endogenous Noncompetitive Inhibitor of Cytochrome P450 1B1 with Enantioselective Activity.

The overexpression of cytochrome P450 1B1 (CYP1B1) is a common characteristic of several diseases and conditions, such as inflammation, cancer, and cardiac hypertrophy. CYP1B1 is believed to contribute to pathogenesis of these diseases by mediating the formation of toxic compounds, either from exogenous or endogenous origin. We recently reported that an arachidonic acid metabolite, 19(S/R-)hydroxyeicosatetraenoic (HETE) acid, protects from cardiac hypertrophy by inhibiting the formation of toxic compounds, midchain HETEs, known to be formed by CYP1B1. This raised the question whether 19(S/R)-HETE can directly inhibit CYP1B1. In the current study, we report that 19(S/R)-HETE enantioselectively inhibits human recombinant CYP1B1 activity measured by 7-ethoxyresorufin O-deethylation assay. 19(S)-HETE is more potent than the R enantiomer (K i = 37.3 and 89.1 nM, respectively). Noncompetitive inhibition was identified as the mechanism of CYP1B1 inhibition, which underlines the potentially important physiologic role of 19(S/R)-HETE as an endogenous CYP1B1 inhibitor; to our knowledge, 19(S/R)-HETE is the first inhibitor of its kind to be reported.

CYP1B1 Activates Wnt/\u03b2-Catenin Signaling through Suppression of Herc5-Mediated ISGylation for Protein Degradation on \u03b2-Catenin in HeLa Cells

Cytochrome P450 1B1 (CYP1B1) acts as a hydroxylase for estrogen and activates potential carcinogens. Moreover, its expression in tumor tissues is much higher than that in normal tissues. Despite this association between CYP1B1 and cancer, the detailed molecular mechanism of CYP1B1 on cancer progression in HeLa cells remains unknown. Previous reports indicated that the mRNA expression level of Herc5, an E3 ligase for ISGylation, is promoted by CYP1B1 suppression using specific small interfering RNA, and that ISGylation may be involved in ubiquitination related to β-catenin degradation. With this background, we investigated the relationships among CYP1B1, Herc5, and β-catenin. RT-PCR and western blot analyses showed that CYP1B1 overexpression induced and CYP1B1 inhibition reduced, respectively, the expression of Wnt/β-catenin signaling target genes including β-catenin and cyclin D1. Moreover, HeLa cells were treated with the CYP1B1 inducer 7,12-dimethylbenz [a] anthracene (DMBA) or the CYP1B1 specific inhibitor, tetramethoxystilbene (TMS) and consequently DMBA increased and TMS decreased β-catenin and cyclin D1 expression, respectively. To determine the correlation between CYP1B1 expression and ISGylation, the expression of ISG15, a ubiquitin-like protein, was detected following CYP1B1 regulation, which revealed that CYP1B1 may inhibit ISGylation through suppression of ISG15 expression. In addition, the mRNA and protein expression levels of Herc5 were strongly suppressed by CYP1B1. Finally, an immunoprecipitation assay revealed a direct physical interaction between Herc5 and β-catenin in HeLa cells. In conclusion, these data suggest that CYP1B1 may activate Wnt/β-catenin signaling through stabilization of β-catenin protein from Herc5-mediated ISGylation for proteosomal degradation.

Cytochrome P450 1B1 inhibition suppresses tumorigenicity of prostate cancer via caspase-1 activation.

Cytochrome P450 1B1 (CYP1B1) is recognized as a universal tumor biomarker and a feasible therapeutic target due to its specific overexpression in cancer tissues. Despite its up-regulation in prostate cancer (PCa), biological significance and clinicopathological features of CYP1B1 are still elusive. Here, we show that overexpression or hyperactivation of CYP1B1 stimulated proliferative, migratory and invasive potential of non-tumorigenic PCa cells. Attenuation of CYP1B1 with its specific small hairpin (sh) RNAs greatly reduced proliferation through apoptotic cell death and impaired migration and invasion in PCa cells. Intratumoral injection of CYP1B1 shRNA attenuated growth of pre-existing tumors. The antitumor effect of CYP1B1 shRNA was also observed in prostate tumor xenograft mouse models. Among the genes altered by CYP1B1 knockdown, reduction of caspase-1 (CASP1) activity attenuated the antitumor effect of CYP1B1 inhibition. Indeed, CYP1B1 regulates CASP1 expression or activity. Finally, CYP1B1 expression was increased in higher grades of PCa and overall survival was significantly reduced in patients with high levels of CYP1B1 protein. CYP1B1 expression was reversely associated with CASP1 expression in clinical tissue samples. Together, our results demonstrate that CYP1B1 regulates PCa tumorigenesis by inhibiting CASP1 activation. Thus, the CYP1B1-CASP1 axis may be useful as a potential biomarker and a therapeutic target for PCa.

The serotonin transporter promotes a pathological estrogen metabolic pathway in pulmonary hypertension via cytochrome P450 1B1.

Pulmonary arterial hypertension (PAH) is a devastating vasculopathy that predominates in women and has been associated with dysregulated estrogen and serotonin signaling. Overexpression of the serotonin transporter (SERT(+)) in mice results in an estrogen-dependent development of pulmonary hypertension (PH). Estrogen metabolism by cytochrome P450 1B1 (CYP1B1) contributes to the pathogenesis of PAH, and serotonin can increase CYP1B1 expression in human pulmonary arterial smooth muscle cells (hPASMCs). We hypothesized that an increase in intracellular serotonin via increased SERT expression may dysregulate estrogen metabolism via CYP1B1 to facilitate PAH. Consistent with this hypothesis, we found elevated lung CYP1B1 protein expression in female SERT(+) mice accompanied by PH, which was attenuated by the CYP1B1 inhibitor 2,3',4,5'-tetramethoxystilbene (TMS). Lungs from female SERT(+) mice demonstrated an increase in oxidative stress that was marked by the expression of 8-hydroxyguanosine; however, this was unaffected by CYP1B1 inhibition. SERT expression was increased in monocrotaline-induced PH in female rats; however, TMS did not reverse PH in monocrotaline-treated rats but prolonged survival. Stimulation of hPASMCs with the CYP1B1 metabolite 16α-hydroxyestrone increased cellular proliferation, which was attenuated by an inhibitor (MPP) of estrogen receptor alpha (ERα) and a specific ERα antibody. Thus, increased intracellular serotonin caused by increased SERT expression may contribute to PAH pathobiology by dysregulation of estrogen metabolic pathways via increased CYP1B1 activity. This promotes PASMC proliferation by the formation of pathogenic metabolites of estrogen that mediate their effects via ERα. Our studies indicate that targeting this pathway in PAH may provide a promising antiproliferative therapeutic strategy.

CYP1B1 inhibition attenuates doxorubicin-induced cardiotoxicity through a mid-chain HETEs-dependent mechanism

Doxorubicin (DOX) has been reported to be a very potent and effective anticancer agent. However, clinical treatment with DOX has been greatly limited due to its cardiotoxicity. Furthermore, several studies have suggested a role for cytochrome P450 1B1 (CYP1B1) and mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) in DOX-induced cardiac toxicity. Therefore, we hypothesized that DOX induced cardiotoxicity is mediated through the induction of CYP1B1 and its associated mid-chain HETEs metabolite. To test our hypothesis, Sprague-Dawley rats and RL-14 cells were treated with DOX in the presence and absence of 2,3′,4,5′-tetramethoxystilbene (TMS), a selective CYP1B1 inhibitor. Thereafter, cardiotoxicity parameters were determined using echocardiography, histopathology, and gene expression. Further, the level of mid-chain HETEs was quantified using liquid chromatography-electron spray ionization-mass spectrometry. Our results showed that DOX induced cardiotoxicity in vivo and in vitro as evidenced by deleterious changes in echocardiography, histopathology, and hypertrophic markers. Importantly, the TMS significantly reversed these changes. Moreover, the DOX-induced cardiotoxicity was associated with a proportional increase in the formation of cardiac mid-chain HETEs both in vivo and in our cell culture model. Interestingly, the inhibition of cardiotoxicity by TMS was associated with a dramatic decrease in the formation of cardiac mid-chain HETEs suggesting a mid-chain HETEs-dependent mechanism. Mechanistically, the protective effect of TMS against DOX-induced cardiotoxicity was mediated through the inhibition of mitogen activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). In conclusion, our study provides the first evidence that the inhibition of CYP1B1 and mid-chain HETE formation attenuate DOX-induced cardiotoxicty.

Abstract 2580: Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells.

Abstract Oral leukoplakia is a precancerous lesion that may progress to squamous cell carcinoma of the head and neck (HNSCC). The long latency period to invasive cancer provides an opportunity for intervention at the precancerous stage. The inability of surgical resection to prevent the recurrence of oral leukoplakia speaks clearly to the need to develop an efficacious regimen for the chemoprevention of this disease. Tobacco smoke constituents and ethanol, the major risk factors for oral leukoplakia and HNSCC, are metabolized by cytochrome P450 1B1 (CYP1B1) to form potentially carcinogenic species. Our previous data indicate that knockdown of CYP1B1 in human oral leukoplakia cells (MSK-Leuk1) by shRNA decreases cell motility and proliferation, relative to control cells overexpressing empty vector. Homoeriodictyol (HED), a flavonoid found in citrus fruits, is a selective inhibitor of CYP1B1 (IC50 0.24 μM). As a next step, we hypothesized that pharmacologic inhibition of CYP1B1 by HED would attenuate cell motility and proliferation in a manner similar to genetic knockdown. Because HED is commonly found in our diet, its toxicity is expected to be low, making it a promising agent for preventive or therapeutic intervention. The ability of HED to inhibit CYP1B1 activity in MSK-Leuk1 cells was established by incubating cells with either vehicle (0.01% ethanol) or HED (5-10 μM) for 72 hrs and quantifying 7-ethoxyresorufin o-deethylation (EROD), a measure of the activity of CYP1B1 and other enzymes of the CYP1 subfamily. HED decreased EROD activity in a dose-dependent manner, with 40% inhibition observed at 10 μM, as compared to vehicle-treated controls. Western blot analyses revealed that CYP1B1 levels were unaltered following HED treatment. The effect of CYP1B1 inhibition on cell motility was examined by exposing MSK-Leuk1 cells to HED (0-10 μM) for 96 hrs followed by a wound-healing assay, in which the ability of cell monolayers to close a gap was monitored for 23 hrs. HED inhibited cell motility in a concentration dependent manner, with maximum inhibition of gap closure reached at 10 μM (6.7 fold relative to vehicle). To assess the effect of CYP1B1 inhibition on cell growth, MSK-Leuk1 cells were exposed to HED (0-10 μM) for 5 days and proliferation was analyzed (Fluorescent DNA Quantitation kit). A direct association was observed between HED dose and inhibition of proliferation, with a 33% decrease observed at 10 μM. In summary, inhibition of CYP1B1 activity by HED resulted in attenuation of the motility and proliferation of cultured MSK-Leuk1 cells. These data confirm that CYP1B1 is a promising molecular target for intervention in HNSCC and provide strong support for the further development of CYP1B1 inhibitors as efficacious agents for the prevention of HNSCC in patients with oral leukoplakia. (Supported by NCI fellowship 1 F32 CA144199-01). Citation Format: Ekaterina G. Shatalova, Margie L. Clapper. Pharmacologic inhibition of cytochrome P450 1B1 attenuates the motility and proliferation of cultured human dysplastic oral leukoplakia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2580. doi:10.1158/1538-7445.AM2013-2580

Induction of apoptosis by a stilbene analog involves bax translocation regulated by p38 MAPK and Akt

trans-Stilbenes have been reported to induce cytochrome P450 1B1 (CYP1B1) inhibition and cell death, however, the molecular mechanisms of the effects are not fully understood. We report here that (1-(2-{3-[2-(2,4-dimethoxy-phenyl)-vinyl]-5-methoxy-phenoxy}ethyl)-1H-imidazole), a synthetic stilbene analog (SA) significantly suppressed TCDD-stimulated CYP1B1 mRNA expression. In HL-60 cells, SA induced apoptosis through activation of p38 MAPK and inactivation of Akt, which in turn activated Bad and mitochondrial death signaling pathway, as evidenced by Bax translocation and cytochrome c release. Expression of dominant negative p38 MAPK or constitutively active Akt significantly prevented cell death and mitochondrial Bax translocation, implicating that p38 MAPK and Akt signaling pathways play crucial roles in stilbene-induced apoptosis of HL-60 cells. These results suggest that SA induces apoptotic cell death as well as CYP1B1 inhibition and may thus be beneficial in cancer prevention.

Modulation of human cytochrome P450 1B1 expression by 2,4,3',5'-tetramethoxystilbene

We have previously shown that 2,4,3',5'-tetramethoxystilbene (TMS), a synthetic trans-stilbene analog, is one of the most potently selective inhibitors of recombinant human cytochrome P450 1B1 (CYP1B1) in vitro. In the present studies, the effects of TMS on CYP1B1 expression were investigated in human cancer cells. TMS significantly inhibited CYP1-mediated 7-ethoxyresorufin O-deethylation activity in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced MCF-7 cells or lung microsomes of Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene. TCDD-stimulated CYP1B1 protein and mRNA expression was significantly suppressed by TMS in a concentration-dependent manner in MCF-7, MCF-10A, and HL-60 cells. Whereas TMS down-regulated TCDD-induced CYP1B1 gene expression, the levels of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator mRNA expression were not changed by TMS treatment. In human cancer cells, TMS induced apoptotic cell death, and the cytotoxic effects of TMS were significant when the cells were incubated with TCDD. CYP1B1 was able to convert TMS to a metabolite(s) when incubated with NADPH. Metabolic activation of TMS by CYP1B1 induced by TCDD may mediate cellular toxicity of TMS in human cancer cells because the sensitivity to TMS in MCF-7 cells treated with TCDD was more significant than in HL-60 cells treated with TCDD. Taken together, our results indicate that TMS acts as a strong modulator of CYP1B1 gene expression as well as a potent selective inhibitor in vitro. The ability of TMS to induce apoptotic cell death in tumor cells, as well as CYP1B1 inhibition, may contribute to its usefulness for cancer chemoprevention.

Lack of correlation between expression of Epstein-Barr virus (EBV) latent membrane protein and bcl-2 oncoprotein in vivo.

To evaluate whether there is any correlation between the expression of Epstein-Barr virus (EBV) latent membrane protein (LMP) and oncoprotein bcl-2 in the lymph node biopsy specimens of a Chinese patient with EBV-related reactive lymphoproliferation who later developed T cell lymphoma after a short period of time. Immunohistochemistry, with a standard alkaline phosphatase antialkaline phosphatase (APAAP) method and New Fuchsin as a chromogen, was used for single staining of bcl-2 or LMP. Double immunostaining combining APAAP and indirect immunofluorescence was performed for dual labelling of LMP and bcl-2. bcl-2 was expressed in 10-30% of cells in the first lymph node biopsy specimen (EBV-associated lymphoproliferative disorder) and 30-50% of cells in the second lymph node biopsy specimen (T cell lymphoma). LMP was expressed in the first biopsy specimen but not in the second. Double immunostaining results showed that around 78% of LMP positive cells were bcl-2 negative and 94% bcl-2 positive cells were LMP negative. Among the very small fraction of LMP and bcl-2 double positive cells, the intensity of bcl-2 staining was heterogeneous and was not always stronger than that observed in LMP negative bcl-2 positive cells. The expression of bcl-2 protein is independent of LMP protein status in vivo. Several mechanisms may be involved in EBV associated lymphomagenesis, and bcl-2 induction may occur independently of LMP expression.