Cytochrome P450 2W1 Research Articles

Morphological predictors of the efficacy of mitotane therapy in adrenocortical cancer

Adrenocortical cancer (ACC) is an orphan malignant tumor of the adrenal cortex with a predominantly poor prognosis and an aggressive clinical course. Nowadays, mitotane is a non-alternative drug in the treatment of ACC. The search for prognostic parameters that determine the sensitivity of ACC to ongoing treatment is currently an urgent task. Expression levels of the large subunit of ribonucleotide reductase M1 (RRM1), cytochrome P450 2W1 (CYP2W1), and sterol- O-acyltransferase-1 (SOAT1) are considered as potential predictors of response to mitotane therapy. To assess the immunohistochemical expression of RRM1, CYP2W1 and SOAT1 in ACC as markers of clinical outcomes and response to the therapy with mitotane. The study included 62 patients older than 17 years of age with a diagnosis of ACC confirmed histologically and immunohistochemically. Mitotane therapy was initiated in 29 patients in the postoperative period, 33 patients were under dynamic observation without concomitant drug treatment. Antibodies to RRM1, CYP2W1, SOAT1 were used diluted in accordance with recommendations of firms-manufacturers for immunohistochemical detection. In the group of patients with low and moderate RRM1, CYP2W1 and SOAT1 immunoreactivity in the tumor and no antitumor therapy, a better DFS was noted (p=0.037, p=0.020 and p=0.001, respectively) compared to the group of patients receiving mitotane therapy at this level of marker expression. With high immunoreactivity of the markers, no statistically significant differences in DFS were found. Consistent with the findings in our study, low expression of RRM1, CYP2W1 and SOAT1 was associated with worse DFS with antitumor therapy. The results of the work indicate the need to assess the levels of immunoreactivity of these markers in patients with ACC before starting treatment with mitotane in order to predict the efficiency of therapy.

Cytochrome P450 Binding and Bioactivation of Tumor-Targeted Duocarmycin Agents.

Duocarmycin natural products are promising anticancer cytotoxins but too potent for systemic use. Re-engineering of the duocarmycin scaffold has enabled the discovery of prodrugs designed for bioactivation by tissue-specific cytochrome P450 (P450) enzymes. Lead prodrugs bioactivated by both P450 isoforms CYP1A1 and CYP2W1 have shown promising results in xenograft studies; however, to fully understand the potential of these agents it is desirable to compare dual-targeting compounds with isoform-selective analogs. Such redesign requires insight into the molecular interactions with these P450 enzymes. Herein binding and metabolism of the individual stereoisomers of the indole-based duocarmycin prodrug ICT2700 and a nontoxic benzofuran analog ICT2726 were evaluated with CYP1A1 and CYP2W1, revealing differences exploitable for drug design. Although enantiomers of both compounds bound to and were metabolized by CYP1A1, the stereochemistry of the chloromethyl fragment was critical for CYP2W1 interactions. CYP2W1 differentially binds the S enantiomer of ICT2726, and its metabolite profile could potentially be used as a biomarker to identify CYP2W1 functional activity. In contrast to benzofuran-based ICT2726, CYP2W1 differentially binds the R isomer of the indole-based ICT2700 over the S stereoisomer. Thus the ICT2700 R configuration warrants further investigation as a scaffold to favor CYP2W1-selective bioactivation. Furthermore, structures of both duocarmycin S enantiomers with CYP1A1 reveal orientations correlating with nontoxic metabolites, and further drug design optimization could lead to a decrease of CYP1A1 bioactivation. Overall, distinctive structural features present in the two P450 active sites can be useful for improving P450-and thus tissue-selective-bioactivation. SIGNIFICANCE STATEMENT: Prodrug versions of the natural product duocarmycin can be metabolized by human tissue-specific cytochrome P450 (P450) enzymes 1A1 and 2W1 to form an ultrapotent cytotoxin and/or high affinity 2W1 substrates to potentially probe functional activity in situ. The current work defines the binding and metabolism by both P450 enzymes to support the design of duocarmycins selectively activated by only one human P450 enzyme.

Cytochrome P450 isoforms 1A1, 1B1 AND 2W1 as targets for therapeutic intervention in head and neck cancer

Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm3) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700.

Cytochrome P450 (CYP) 2W1 affect steroid secretion in adrenocortical cell line and tumor tissues

Cytochrome P450 (CYP) 2W1 affect steroid secretion in adrenocortical cell line and tumor tissues

Effects of Germline CYP2W1*6 and CYP2B6*6 Single Nucleotide Polymorphisms on Mitotane Treatment in Adrenocortical Carcinoma: A Multicenter ENSAT Study

Mitotane is the only approved drug for advanced adrenocortical carcinoma (ACC) and no biomarkers are available to predict attainment of therapeutic plasma concentrations and clinical response. Aim of the study was to evaluate the suitability of cytochrome P450(CYP)2W1 and CYP2B6 single nucleotide polymorphisms (SNPs) as biomarkers. A multicenter cohort study including 182 ACC patients (F/M = 121/61) treated with mitotane monotherapy after radical resection (group A, n = 103) or in not completely resectable, recurrent or advanced disease (group B, n = 79) was performed. CYP2W1*2, CYP2W1*6, CYP2B6*6 and CYP2B6 rs4803419 were genotyped in germline DNA. Mitotane blood levels were measured regularly. Response to therapy was evaluated as time to progression (TTP) and disease control rate (DCR). Among investigated SNPs, CYP2W1*6 and CYP2B6*6 correlated with mitotane treatment only in group B. Patients with CYP2W1*6 (n = 21) achieved less frequently therapeutic mitotane levels (>14 mg/L) than those with wild type (WT) allele (76.2% vs 51.7%, p = 0.051) and experienced shorter TTP (HR = 2.10, p = 0.019) and lower DCR (chi-square = 6.948, p = 0.008). By contrast, 55% of patients with CYP2B6*6 vs. 28.2% WT (p = 0.016) achieved therapeutic range. Combined, a higher rate of patients with CYP2W1*6WT+CYP2B6*6 (60.6%) achieved mitotane therapeutic range (p = 0.034). In not completely resectable, recurrent or advanced ACC, CYP2W1*6 SNP was associated with a reduced probability to reach mitotane therapeutic range and lower response rates, whereas CYP2B6*6 correlated with higher mitotane levels. The association of these SNPs may predict individual response to mitotane.

Micro RNA-4651 Serves as a Potential Biomarker for Prognosis When Selecting Hepatocellular Carcinoma Patients for Postoperative Adjuvant Transarterial Chemoembolization Therapy.

Our previous reports have shown that microRNA‐4651 is a potential early diagnostic and prognostic marker for hepatocellular carcinoma. We aimed to investigate whether microRNA‐4651 modified postoperative adjuvant transarterial chemoembolization (pa‐TACE) to improve the prognosis of hepatocellular carcinoma. A hospital‐based retrospective study, including 302 patients with advanced‐stage hepatocellular carcinoma who received tumor resection or tumor resection plus pa‐TACE as an initial therapy, was conducted to assess the effects of microRNA‐4651 on pa‐TACE treatment. MicroRNA‐4651 expression in tumor tissues was tested using the TaqMan‐PCR technique. The sensitivity of tumor cells to doxorubicin (an anticancer drug used in pa‐TACE procedure) was analyzed by the half‐maximal inhibitory concentration (IC50). Upregulated microRNA‐4651 expression in tumor tissues can improve the therapeutic response of patients with hepatocellular carcinoma on pa‐TACE (hazard ratios [95% confidence intervals] = 0.32 [0.22‐0.46] for death risk and 0.39 [0.28‐0.56] for tumor‐recurrence risk, respectively), but downregulated expression cannot. Functional analyses–displayed microRNA‐4651 mimics decreased while its inhibitor increased the IC50 of tumor cells to doxorubicin (0.65 [0.61‐0.69] versus 2.17 [1.98‐2.37] µM). Cytochrome P450 2W1 was shown as a possible target of microRNA‐4651. Additionally, dysregulation of microRNA‐4651 also affected the clinical pathological features of hepatocellular carcinoma and was an independent prognostic factor for this cancer. Conclusion: These results indicate that increasing microRNA‐4651 expression may be beneficial for pa‐TACE in improving hepatocellular carcinoma prognosis.

Abstract 5234: Expression of cytochrome P450 in head and neck cancer cell lines

Abstract Background: The high expression of cytochrome P450 (CYP) enzymes including isoforms CYP1A1, 1B1 and 2W1 in some tumor types compared to normal tissue make these potential targets for targeted drug discovery. Our efforts in this space have resulted in the discovery of duocarmycin bioprecursors that are bioactivated by CYP1A1 and CYP2W1 expressed in malignant tissues1-2. Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including CYPs to detoxify or activate such carcinogens. Here, we report on the expression of CYP1A1, CYP1B1 and CYP2W1 in HNC cell lines and human primary tissues. Materials and methods: A panel of HNC cell lines (FaDu, Detroit-562, A253, OSC19) was characterized for CYP1A1, CYP1B1 and CYP2W1 gene expression using real time PCR. Furthermore, immunofluorescence (IF) was used to detect protein expression in HNC cell lines and immunohistochemistry (IHC) in xenografts, paraffin-embedded and snap-frozen human HNC tissue. Anti-proliferative activity was measured using the MTT assay and DNA damage was measured using the comet assay. Results: In the HNC cell lines significant expression (5-10 fold) was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit xenografts and in a small cohort of 10 human HNC samples as measured by IF and IHC, respectively. In agreement with CYP1 expression, the cytotoxicity results of ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines suggesting bioactivation of these two lead duocarmycin bioprecursors. Conclusion: Accordingly, from these data we can conclude that CYP1A1, CYP1B1 and CYP2W1 are expressed in selected HNC cell lines and tumor tissue. In addition, early evidence suggests that CYP2W1 expression is stimulated by the presence of an in vivo microenvironment. However a larger cohort (≥ 60) of clinical HNC samples will be used to confirm this observation and will be reported at the annual AACR meeting. [1] Sheldrake et al.Reengineering of the duocarmycin structural architecture enables bioprecursors development targeting CYP1A1 and CYP2W1 for biological activity. J Med Chem. 2013, 56, 62737. [2] Travica et al. Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins. Clin Cancer Res. 2013, 19, 295261. Citation Format: Daniela F. Presa, Goreti Ribeiro Morais, Mark Sutherland, Jim McCaul, Jacques Robert, Laurence Patterson, Klaus Pors. Expression of cytochrome P450 in head and neck cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5234. doi:10.1158/1538-7445.AM2017-5234

Human Cytochrome P450 2W1 Is Not Expressed in Adrenal Cortex and Is Only Rarely Expressed in Adrenocortical Carcinomas.

Human cytochome P450 2W1 (CYP2W1) enzyme is expressed in fetal colon and in colon tumors. The level of expression is higher in colon metastases than in the parent tumors and the enzyme is a possible drug target for treatment of colorectal cancer, as demonstrated in mouse xenograft studies. A previous study published in this journal reported that CYP2W1 is highly expressed in normal and transformed adrenal tissue. However, adrenal expression of CYP2W1 protein was not seen in previous studies in our research group. To clarify this inconsistency, we have used qRT-PCR and Western blotting with CYP2W1-specific antibodies to probe a panel of 27 adrenocortical carcinomas and 35 normal adrenal cortex samples. CYP2W1 mRNA expression is seen in all samples. However, significant CYP2W1 protein expression was found in only one tumor sample (a testosterone-producing adrenocortical carcinoma) and not in any normal tissue. Differences in the specificity of the CYP2W1 antibodies used in the two studies may explain the apparent discrepancy. We conclude that normal adrenal tissue lacks P450 2W1 enzyme expression; also, adrenocortical carcinomas generally do not express the enzyme. This information thus underline the colon cancer specificity of CYP2W1 enzyme expression and has implications for the development of anti-colon cancer therapies based on CYP2W1 as a drug target, since 2W1-dependent bioactivation of prodrugs for CYP2W1 will not take place in normal adrenal tissue or other non-transformed tissues.

Abstract 3105: Probing cytochrome P450 activity with benzofuran-based duocarmycins in a panel of head and neck cancer cell lines and CYP1A1, 1B1 and CYP2W1 transfected cell lines

Abstract Background: The cytochrome P450 (CYP) isoforms CYP1A1, 1B1 and 2W1 are highly expressed in tumour tissue and surrounding stroma compared to nearby normal tissue, which provides an opportunity for development of selective cancer therapeutics as previously reported by us.1-2 However, no reagent is available to interrogate CYP2W1 and hence the purpose of this study was to evaluate a series of novel benzofuran-based duocarmycins as chemical probes to study the functional activity of CYP2W1. Materials and methods: Standard materials and methods can be found in the references below. Briefly, these include route to synthesis of duocarmycin bioprecursors, use of recombinant CYP bactosomes with LC/MS for metabolite identification, and cell culture MTT proliferation assay. CYP expression was measured by RT-PCR and western blot. Results: A panel of head and neck cancer (HNC) cell lines (SCC4, FaDu, Detroit-562, A-253, OSC19, UT-SCC5, UT-SCC10, UT-SCC14) were analysed for the expression of CYP1A1, CYP1B1 and CYP2W1. Significant expression (5-10 fold) was shown at the gene level while protein expression experiments are currently underway and will be reported at the meeting. Furthermore, CYP2W1 functional activity was probed with a novel series of benzofuran-based duocarmycin analogues. The compounds were incubated with recombinant CYP bactosomes or HEK2932W1-transfected cells and their oxidative metabolism was studied using LCMS. CYP2W1 was shown to metabolise one chemical probe, ICT2726, to a unique metabolite not observed for any other CYP isoform. The compounds were investigated for antiproliferative activity in a panel of cell lines (CHO, CHO1A1, RT112, HEK293, HEK2932W1, Cal27, Cal271B1, Cal33, Cal331B1). Obtained IC50 values > 1 μM, which for these ultrapotent (picomolar) duocarmycins, indicated no CYP-bioactivation, consistent with no active duocarmycin metabolite identified from the LCMS studies. Conclusion: Our findings support the use of non-toxic ICT2726 as a chemical tool that can be employed in vitro to probe CYP2W1 functional activity. [1] Sheldrake et al. Re-engineering of the duocarmycin structural architecture enables bioprecursor development targeting CYP1A1 and CYP2W1 for biological activity. J Med Chem. 2013, 56, 6273-7. [2] Travica et al. Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins. Clin Cancer Res. 2013, 19, 2952-61. Citation Format: Daniela Presa, Paul M. Loadman, Giuliano Nigro, Helen M. Sheldrake, Mark Sutherland, Steven D. Shnyder, Valerie Le Morvan, Jacques Robert, Magnus Ingelman-Sundberg, Laurence H. Patterson, Klaus Pors. Probing cytochrome P450 activity with benzofuran-based duocarmycins in a panel of head and neck cancer cell lines and CYP1A1, 1B1 and CYP2W1 transfected cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3105.

Membrane topology and search for potential redox partners of colon cancer‐specific cytochrome P450 2W1

Cytochrome P450 2W1 (CYP2W1) is a colon tumor-specific enzyme, suggested as a potential target for cancer therapy. In contrast to other endoplasmic reticulum P450s, we found completely inverted ER membrane topology of CYP2W1 using different approaches (redox sensitive luciferase assay and protease protection assay) and demonstrated that canonical CYP reductants, cytochrome P450 reductase, and cytochrome b5 cannot serve as electron donors for CYP2W1. Moreover, the reduced catalytic activity of the Asn177 mutant that is modified by glycan moieties in the wild-type enzyme indicates a functional relevance of CYP2W1 glycosylation.

Cytochrome P450 2W1 (CYP2W1) in Colorectal Cancers.

Cytochrome P450, family 2, subfamily W, polypeptide 1 (CYP2W1) is a newly identified monooxygenase enzyme that is expressed specifically in tumor tissues and during fetal life. Particularly, high expression of CYP2W1 was observed in up to 60% of colorectal cancers and its expression correlated with poor survival. CYP2W1 has been shown to metabolize various endogenous substrates including lysophospholipids and several procarcinogens, such as polycyclic aromatic hydrocarbon. The specific substrate for CYP2W1, however, is currently unknown. Due to its tumor-specific expression and its unique catalytic activities in colorectal cancers, CYP2W1 was deemed as an interesting target in colorectal cancer therapy. This review sought to summarize the current understanding of the CYP2W1 biology and biochemistry, its genetic polymorphisms and cancer risk, and its implication as a tumor-specific diagnostic and therapeutic target.

Novel single nucleotide polymorphisms (SNPs) of CYP2W1 gene in Chinese Uygur and Han populations

Cytochrome P450 2W1 (CYP2W1) is expressed specially in certain cancers and could metabolize some substances into cytotoxic antitumor drugs in Caucasian ethnic population. To investigate the genetic polymorphism of CYP2W1 in Chinese, all the nine exons and exon–intron junctions were sequenced by dideoxy chain termination method among 385 Chinese subjects (including 223 Han and 162 Uygur). The present results showed that 40 single nucleotide polymorphisms (SNPs) were detected (14 in the exons and 26 in the introns), 10 were novel variants of which in Chinese. There were 7 novel SNPs in the exons and other 3 novel SNPs in the introns. Four of the 6 novel non-synonymous variations in exons, 131T > C (Leu44Pro), 1289C > A (Ala88Glu), 2027G > A (Arg187Gln) and 5070C > T (Thr383Met) were computationally predicted to affect CYP2W1 protein function, in spite of these variants were heterozygotes. Moreover, the allele frequencies in 6 known SNPs including CYP2W1*2 (2008G > A) and CYP2W1*3 (173A > C) were analyzed, which were significantly lower in Chinese Han (2.9% and 0.0%, respectively) and Uygur (5.2% and 0.0%, respectively) individuals, than those reported previously in Caucasians (9.1% and 33.1%, respectively, P < 0.05). These data provide useful information on the pharmacogenetic studies of CYP2W1 among Chinese and other ethnic populations.

Abstract 4541: Tumor-selective bioactivation of duocarmycin bioprecursors by cytochrome P450 enzymes provides an opportunity to treat drug-resistant breast cancer cells

Abstract Background The cytochrome P450 (CYP) enzymes are responsible for the oxidation of a diverse range of xenobiotic and endogenous compounds. The high expression of CYP1A1, 1B1 and 2W1 in tumour tissue and surrounding stroma compared to nearby normal tissue provides an opportunity for development of selective cancer therapeutics. The ultrapotent duocarmycins are ideal candidates for bioprecursor development and we have demonstrated that these can be re-engineered into derivatives selectively activated by CYP1A1, 1B1 (unpublished) and 2W1 in vitro and in vivo.1-3 Materials and methods Standard material and methods can be found in the references below. Briefly, these include synthetic chemistry necessary for synthesis of duocarmycin bioprecursors, use of recombinant CYP bactosomes for metabolite identification using LC/MS and cell culture experiments using the MTT assay. Results In the present study, we have focussed on the potential of eradicating both sensitive and drug-resistant breast cancer cells (MCF-7, MDA-MB-468 and MDA-MB-231). We have evaluated a library of novel compounds in vitro and shown 3-modified CPI bioprecursors to be bioactivated to potent (&amp;lt;10 nM) metabolites capable of damaging DNA as shown using H2A.X phosphorylation as a marker of DNA damage. LCMS confirmed the presence of hydroxylated seco-duocarmycins and structure-activity relationship studies revealed that subtle changes to the substitution pattern of the DNA recognition motif resulted in changes to the CYP bioactivation and chemosensitivity. The duocarmycin bioprecursors were intolerant by functionalization at position 7, to some extent position 6 while position 5 was needed for potent bioactivation and anticancer activity. Conclusion Modifications to the structure of the seco-duocarmycins influences the extent of their CYP-mediated activation and indicates their potential for breast cancer chemotherapy. [1] Sheldrake et al. Re-engineering of the duocarmycin structural architecture enables bioprecursor development targeting CYP1A1 and CYP2W1 for biological activity. J Med Chem. 2013, 56, 6273-7. [2] Travica et al. Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins. Clin Cancer Res. 2013, 19, 2952-61. [3] Pors et al. Modification of the duocarmycin pharmacophore enables CYP1A1 targeting for biological activity. Chem Commun. 2011, 47, 12062-4. Citation Format: Steven D. Shnyder, Paul M. Loadman, Mark Sutherland, Helen M. Sheldrake, Mark Searcey, Laurence H. Patterson, Klaus Pors. Tumor-selective bioactivation of duocarmycin bioprecursors by cytochrome P450 enzymes provides an opportunity to treat drug-resistant breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4541. doi:10.1158/1538-7445.AM2015-4541

Developmental regulation and induction of cytochrome P450 2W1, an enzyme expressed in colon tumors.

Cytochrome P450 2W1 (CYP2W1) is expressed predominantly in colorectal and also in hepatic tumors, whereas the levels are insignificant in the corresponding normal human adult tissues. CYP2W1 has been proposed as an attractive target for colorectal cancer (CRC) therapy by exploiting its ability to activate duocarmycin prodrugs to cytotoxic metabolites. However, its endogenous function, regulation and developmental pattern of expression remain unexplored. Here we report the CYP2W1 developmental expression in the murine and human gastrointestinal tissues. The gene expression in the colon and small intestine commence at early stages of embryonic life and is completely silenced shortly after the birth. Immunohistochemical analysis of human fetal colon revealed that CYP2W1 expression is restricted to the crypt cells. The silencing of CYP2W1 after birth correlates with the increased methylation of CpG-rich regions in both murine and human CYP2W1 genes. Analysis of CYP2W1 expression in the colon adenocarcinoma cell line HCC2998 revealed that the gene expression can be induced by e.g. the antitumor agent imatinib, linoleic acid and its derivatives. The imatinib mediated induction of CYP2W1 suggests an adjuvant therapy to treatment with duocarmycins that thus would involve induction of tumor CYP2W1 levels followed by the CYP2W1 activated duocarmycin prodrugs. Taken together these data strongly support further exploration of CYP2W1 as a specific drug target in CRC.

Abstract 1779: CYP2W1 as a novel therapeutic target in colon cancer

Abstract Colorectal cancer is the third most frequently diagnosed malignancy in both men and women and a leading cause of cancer-related deaths in the Western world. Advances in neoadjuvant/adjuvant chemotherapy (including 5-fluorouracil, irinotecan, and oxaliplatin) as well as radiation therapy have improved the clinical outcome, however, the overall impact of these amended treatment regiments has been relatively modest, leaving metastasised or recurrent disease largely incurable by conventional approaches and requiring the development of better therapeutics. Targeted chemotherapy aimed towards more effective advanced colon cancer treatment could be achieved by the use of agents that are systemically inactive but selectively converted to potent cytotoxins locoregionally. We have shown cytochrome P450 2W1 (CYP2W1) to be overexpressed in colorectal cancer, and recently shown this enzyme to be a druggable target in a proof-of-concept study using CYP2W1-transfected colon cancer cells [selected references below]. At this meeting we will present an update on our progress on re-engineering the duocarmycin family of compounds for CYP2W1-targeting. New findings reveal that subtle changes in the duocarmycin pharmacophore can lead to dramatic changes (&amp;gt; 100-fold) in the anti-cancer activity. This drop in anticancer activity of small molecules such as ICT2726 means these can be used as biomarker to detect CYP2W1 functional activity, which can be used alongside immunological methods to characterise malignant colorectal tissue. Similarly, interrogation of R and S-enantiomers of duocarmycin bioprecursors also reveal significant differential anticancer activity in vitro. Studies are currently underway to understand if this differential activity is also observed in CYP2W1-expressing colon cancer xenografts and the results will be presented at the AACR meeting. Our findings reveal the opportunities in targeting CYP2W1 as a novel therapeutic approach in colon cancer chemotherapy. [1] Travica S. et al. Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins. Clin. Cancer Res. 2013, 19(11), 2952-61. [2] Sheldrake et al. Re-engineering of the Duocarmycin Structural Architecture Enables Bioprecursor Development Targeting CYP1A1 and CYP2W1 for Biological Activity. J Med Chem. 2013, 56 (15), 6273-7. [3] Stenstedt, K. et al. Cytochrome P450 2W1 polymorphism: functional aspects and relation to risk for colorectal cancer. Pharmacogenomics. 2013, 14(13), 1615-1622. [4] Sutherland M. et al. Anti-tumor Activity of a Duocarmycin Analogue Rationalised to be Metabolically Activated By Cytochrome P450 1A1 in Human Bladder Cancer. Mol Cancer Ther. 2013, 12 (1), 27-37. [5] Pors K. et al. Modification of the duocarmycin pharmacophore enables CYP1A1 targeting for biological activity. Chem. Commun., 2011, 47, 12062-4. [6] Stenstedt K et al. The expression of CYP2W1: a prognostic marker in colon cancer. Anticancer Res. 2012, 32, 3869-74. Citation Format: Klaus Pors, Paul M. Loadman, Sandra Travica, Steven D. Shnyder, Mark Sutherland, Helen Sheldrake, Mark Searcey, Inger Johansson, Souren Mkrtchian, Magnus Ingelman-Sundberg, Laurence H. Patterson. CYP2W1 as a novel therapeutic target in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1779. doi:10.1158/1538-7445.AM2014-1779

Expression of CYP2W1 in the adrenal gland: relationship with hormone secretion and clinical outcome

Adrenocortical tumors consist of frequent adenomas (ACA) and rare highly malignant carcinomas (ACC). The orphan enzyme cytochrome P450 2W1 (CYP2W1) is selectively expressed in some cancers and represent a promising new drug target in cancer therapy.

Colon Cancer–Specific Cytochrome P450 2W1 Converts Duocarmycin Analogues into Potent Tumor Cytotoxins

Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents. The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts. Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1.

Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids

Human cytochrome P450 (P450) 2W1 is still considered an "orphan" because its physiological function is not characterized. To identify its substrate specificity, the purified recombinant enzyme was incubated with colorectal cancer extracts for untargeted substrate searches using an LC/MS-based metabolomic and isotopic labeling approach. In addition to previously reported fatty acids, oleyl (18:1) lysophosphatidylcholine (LPC, lysolecithin) was identified as a substrate for P450 2W1. Other human P450 enzymes tested showed little activity with 18:1 LPC. In addition to the LPCs, P450 2W1 acted on a series of other lysophospholipids, including lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylethanolamine, and lysophosphatidic acid but not diacylphospholipids. P450 2W1 utilized sn-1 18:1 LPC as a substrate much more efficiently than the sn-2 isomer; we conclude that the sn-1 isomers of lysophospholipids are preferred substrates. Chiral analysis was performed on the 18:1 epoxidation products and showed enantio-selectivity for formation of (9R,10S) over (9S,10R). [corrected]. The kinetics and position specificities of P450 2W1-catalyzed oxygenation of lysophospholipids (16:0 LPC and 18:1 LPC) and fatty acids (C16:0 and C18:1) were also determined. Epoxidation and hydroxylation of 18:1 LPC are considerably more efficient than for the C18:1 free fatty acid.

Colorectal Cancer-Specific Cytochrome P450 2W1: Intracellular Localization, Glycosylation, and Catalytic Activity

Cytochrome P450 2W1 (CYP2W1) is expressed at high levels in colorectal cancer cells. Moreover, we have shown previously that a higher tumor expression is associated with less survival. In this study, we characterize post-translational modification, inverted endoplasmic reticulum (ER) topology, and catalytic activity of CYP2W1. The analysis of colorectal normal and cancer tissues and CYP2W1 overexpressing human embryonic kidney (HEK) 293 cells showed that a fraction of CYP2W1 is modified by N-glycosylation. Bioinformatic analysis identified Asn177 as the only possible glycosylation site of CYP2W1, which was supported by the inability of an N177A mutant to be glycosylated in HEK 293 cells. Analysis of the membrane topology indicated that unlike other cytochromes P450, CYP2W1 in HEK 293-transfected cells and in nontransfected Caco2TC7 and HepG2 cells is oriented toward the lumen of the ER, a topology making CYP2W1 available to the ER glycosylation machinery. Immunofluorescence microscopy and cell surface biotinylation experiments revealed approximately 8% of the CYP2W1 on the cell surface. Despite the reverse orientation of CYP2W1 in the ER membrane, apparently making functional interactions with NADPH-cytochrome P450 reductase impossible, CYP2W1 in HEK 293 cells was active in the metabolism of indoline substrates and was able to activate aflatoxin B1 into cytotoxic products. The study identifies for the first time a cytochrome P450 enzyme with a luminal ER orientation and still retaining catalytic activity. Together, these results suggest the possibility of using CYP2W1 as a drug target in the treatment of colon cancer using antibodies and/or specific CYP2W1 activated prodrugs.

Molecular modeling of human cytochrome P450 2W1 and its interactions with substrates

The human cytochrome P450 2W1 (CYP2W1) was categorized into the so-called “orphan” CYPs because of its unknown enzymatic function. However, recent studies showed that the recombinant CYP2W1 exhibited broad catalytic activity towards several chemicals. Furthermore, this enzyme was selectively expressed in some forms of cancers, whereas a very low expression was found in human normal issues. These render CYP2W1 as a potential drug target for cancer therapy. At present, however, little information is available on the active site topology and the substrate binding modes of CYP2W1. In this study, the three-dimensional model of CYP2W1 was constructed using the homology modeling method. Two known substrates, benzphetamine and indole, were then docked into the active site, and refined by molecular dynamics simulations. The interaction energy between the substrates and the enzyme was calculated and analyzed by using the MM-GBSA method. The results indicated that the constructed CYP2W1 model can account for the regioselectivity of this enzyme towards the known substrates and van der Waals interactions were the driving force for the substrate binding. Several key residues were identified to be responsible for the binding of indole and benzphetamine with CYP2W1. These findings provide useful information for the detailed characterization of the biological roles of CYP2W1 and structure-based drug design of this enzyme.