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4 Ipomeanol

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Katharina Roellecke – One of the best experts on this subject based on the ideXlab platform.

  • Influence of Stereochemistry on the Bioactivation and Glucuronidation of 4Ipomeanol
    The Journal of pharmacology and experimental therapeutics, 2018
    Co-Authors: Aaron M. Teitelbaum, Katharina Roellecke, Oliver T. Parkinson, Dale Whittington, Matthew G. Mcdonald, John P. Kowalski, Michele Scian, Helmut Hanenberg, Constanze Wiek, Allan E. Rettie

    Abstract:

    A potential CYP4B1 suicide gene application in engineered T-cell treatment of blood cancers has revived interest in the use of 4Ipomeanol (IPO) in gene-directed enzyme prodrug therapy, in which disposition of the administered compound may be critical. IPO contains one chiral center at the carbon bearing a secondary alcohol group; it was of interest to determine the effect of stereochemistry on 1) CYP4B1-mediated bioactivation and 2) (UGT)-mediated glucuronidation. First, (R)-IPO and (S)-IPO were synthesized and used to assess cytotoxicity in HepG2 cells expressing rabbit CYP4B1 and re-engineered human CYP4B1, where the enantiomers were found to be equipotent. Next, a sensitive UPLC-MS/MS assay was developed to measure the IPO-glucuronide diastereomers and product stereoselectivity in human tissue microsomes. Human liver and kidney microsomes generated (R)- and (S)-IPO-glucuronide diastereomers in ratios of 57:43 and 79:21, respectively. In a panel of 13 recombinantly expressed UGTs, UGT1A9 and UGT2B7 were the major isoforms responsible for IPO glucuronidation. (R)-IPO-glucuronide diastereoselectivity was apparent with each recombinant UGT, except UGT2B15 and UGT2B17, which favored the formation of (S)-IPO-glucuronide. Incubations with IPO and the UGT1A9-specific chemical inhibitor niflumic acid significantly decreased glucuronidation in human kidney, but only marginally in human liver microsomes, consistent with known tissue expression patterns of UGTs. We conclude that IPO glucuronidation in human kidney is mediated by UGT1A9 and UGT2B7. In human liver, it is mediated primarily by UGT2B7 and, to a lesser extent, UGT1A9 and UGT2B15. Overall, the lack of pronounced stereoselectivity for IPO’s bioactivation in CYP4B1-transfected HepG2 cells, or for hepatic glucuronidation, suggests the racemate is an appropriate choice for use in suicide gene therapies.

  • Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells.
    Protein engineering design & selection : PEDS, 2017
    Co-Authors: Katharina Roellecke, Allan E. Rettie, John P. Kowalski, Helmut Hanenberg, Constanze Wiek, Vera D. Jäger, Veselin H Gyurov, Stephanie Mielke, Marco Girhard

    Abstract:

    Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4Ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wild-type rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l-1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

  • optimized human cyp4b1 in combination with the alkylator prodrug 4 Ipomeanol serves as a novel suicide gene system for adoptive t cell therapies
    Gene Therapy, 2016
    Co-Authors: Katharina Roellecke, E L Virts, R Einholz, K Z Edson, Bianca Altvater, Claudia Rossig, Dorothee Von Laer, K Scheckenbach, Martin Wagenmann, Dirk Reinhardt

    Abstract:

    Optimized human CYP4B1 in combination with the alkylator prodrug 4Ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies

Allan E. Rettie – One of the best experts on this subject based on the ideXlab platform.

  • Structure-Activity Relationships for CYP4B1 Bioactivation of 4Ipomeanol Congeners: Direct Correlation between Cytotoxicity and Trapped Reactive Intermediates.
    Chemical research in toxicology, 2019
    Co-Authors: John P. Kowalski, Dale Whittington, Matthew G. Mcdonald, Michele Scian, Helmut Hanenberg, Constanze Wiek, Miklos Guttman, Marco Girhard, Allan E. Rettie

    Abstract:

    Cytochrome P450 4B1 (CYP4B1) has been explored as a candidate enzyme in suicide gene systems for its ability to bioactivate the natural product 4Ipomeanol (IPO) to a reactive species that causes cytotoxicity. However, metabolic limitations of IPO necessitate discovery of new “pro-toxicant” substrates for CYP4B1. In the present study, we examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells expressing CYP4B1. This compound series maintains the furan warhead of IPO while replacing its alcohol group with alkyl chains of varying length (C1-C8). Compounds with C3-C6 carbon chain lengths showed similar potency to IPO (LD50 ≈ 5 μM). Short chain analogs ( 6 carbons) exhibited reduced toxicity, resulting in a parabolic relationship between alkyl chain length and cytotoxicity. A similar parabolic relationship was observed between alkyl chain length and reactive intermediate formation upon trapping of the putative enedial as a stable pyrrole adduct in incubations with purified recombinant rabbit CYP4B1 and common physiological nucleophiles. These parabolic relationships reflect the lower affinity of shorter chain compounds for CYP4B1 and increased ω-hydroxylation of the longer chain compounds by the enzyme. Furthermore, modest time-dependent inhibition of CYP4B1 by N-pentyl-3-furancarboxamide was completely abolished when trapping agents were added, demonstrating escape of reactive intermediates from the enzyme after bioactivation. An insulated CYP4B1 active site may explain the rarely observed direct correlation between adduct formation and cell toxicity reported here.

  • Influence of Stereochemistry on the Bioactivation and Glucuronidation of 4Ipomeanol
    The Journal of pharmacology and experimental therapeutics, 2018
    Co-Authors: Aaron M. Teitelbaum, Katharina Roellecke, Oliver T. Parkinson, Dale Whittington, Matthew G. Mcdonald, John P. Kowalski, Michele Scian, Helmut Hanenberg, Constanze Wiek, Allan E. Rettie

    Abstract:

    A potential CYP4B1 suicide gene application in engineered T-cell treatment of blood cancers has revived interest in the use of 4Ipomeanol (IPO) in gene-directed enzyme prodrug therapy, in which disposition of the administered compound may be critical. IPO contains one chiral center at the carbon bearing a secondary alcohol group; it was of interest to determine the effect of stereochemistry on 1) CYP4B1-mediated bioactivation and 2) (UGT)-mediated glucuronidation. First, (R)-IPO and (S)-IPO were synthesized and used to assess cytotoxicity in HepG2 cells expressing rabbit CYP4B1 and re-engineered human CYP4B1, where the enantiomers were found to be equipotent. Next, a sensitive UPLC-MS/MS assay was developed to measure the IPO-glucuronide diastereomers and product stereoselectivity in human tissue microsomes. Human liver and kidney microsomes generated (R)- and (S)-IPO-glucuronide diastereomers in ratios of 57:43 and 79:21, respectively. In a panel of 13 recombinantly expressed UGTs, UGT1A9 and UGT2B7 were the major isoforms responsible for IPO glucuronidation. (R)-IPO-glucuronide diastereoselectivity was apparent with each recombinant UGT, except UGT2B15 and UGT2B17, which favored the formation of (S)-IPO-glucuronide. Incubations with IPO and the UGT1A9-specific chemical inhibitor niflumic acid significantly decreased glucuronidation in human kidney, but only marginally in human liver microsomes, consistent with known tissue expression patterns of UGTs. We conclude that IPO glucuronidation in human kidney is mediated by UGT1A9 and UGT2B7. In human liver, it is mediated primarily by UGT2B7 and, to a lesser extent, UGT1A9 and UGT2B15. Overall, the lack of pronounced stereoselectivity for IPO’s bioactivation in CYP4B1-transfected HepG2 cells, or for hepatic glucuronidation, suggests the racemate is an appropriate choice for use in suicide gene therapies.

  • Ligand characterization of CYP4B1 isoforms modified for high-level expression in Escherichia coli and HepG2 cells.
    Protein engineering design & selection : PEDS, 2017
    Co-Authors: Katharina Roellecke, Allan E. Rettie, John P. Kowalski, Helmut Hanenberg, Constanze Wiek, Vera D. Jäger, Veselin H Gyurov, Stephanie Mielke, Marco Girhard

    Abstract:

    Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4Ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wild-type rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l-1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

Dirk Reinhardt – One of the best experts on this subject based on the ideXlab platform.

  • optimized human cyp4b1 in combination with the alkylator prodrug 4 Ipomeanol serves as a novel suicide gene system for adoptive t cell therapies
    Gene Therapy, 2016
    Co-Authors: Katharina Roellecke, E L Virts, R Einholz, K Z Edson, Bianca Altvater, Claudia Rossig, Dorothee Von Laer, K Scheckenbach, Martin Wagenmann, Dirk Reinhardt

    Abstract:

    Optimized human CYP4B1 in combination with the alkylator prodrug 4Ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies

  • Optimized human CYP4B1 in combination with the alkylator prodrug 4Ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies
    Gene Therapy, 2016
    Co-Authors: Katharina Roellecke, E L Virts, R Einholz, K Z Edson, Bianca Altvater, Claudia Rossig, K Scheckenbach, Martin Wagenmann, D Von Laer, Dirk Reinhardt

    Abstract:

    Engineering autologous or allogeneic T cells to express a suicide gene can control potential toxicity in adoptive T-cell therapies. We recently reported the development of a novel human suicide gene system that is based on an orphan human cytochrome P450 enzyme, CYP4B1, and the naturally occurring alkylator prodrug 4Ipomeanol. The goal of this study was to systematically develop a clinically applicable self-inactivating lentiviral vector for efficient co-expression of CYP4B1 as an ER-located protein with two distinct types of cell surface proteins, either MACS selection genes for donor lymphocyte infusions after allogeneic stem cell transplantation or chimeric antigen receptors for retargeting primary T cells. The U3 region of the myeloproliferative sarcoma virus in combination with the T2A site was found to drive high-level expression of our CYP4B1 mutant with truncated CD34 or CD271 as MACS suitable selection markers. This lentiviral vector backbone was also well suited for co-expression of CYP4B1 with a codon-optimized CD19 chimeric antigen receptor (CAR) construct. Finally, 4Ipomeanol efficiently induced apoptosis in primary T cells that co-express mutant CYP4B1 and the divergently located MACS selection and CAR genes. In conclusion, we here developed a clinically suited lentiviral vector that supports high-level co-expression of cell surface proteins with a potent novel human suicide gene.