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Research ArticleSpecial Section on Emerging Novel Enzyme Pathways in Drug Metabolism

Discovery of a Novel Microsomal Epoxide Hydrolase–Catalyzed Hydration of a Spiro Oxetane

Xue-Qing Li, Martin A. Hayes, Gunnar Grönberg, Kristina Berggren, Neal Castagnoli Jr. and Lars Weidolf
Drug Metabolism and Disposition August 2016, 44 (8) 1341-1348; DOI: https://doi.org/10.1124/dmd.116.071142
Xue-Qing Li
Cardiovascular and Metabolic Diseases(X.-Q.L., M.A.H., L.W.) and Respiratory, Inflammation, and Autoimmune Disease (G.G., K.B.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; and Department of Chemistry, Virginia Tech, Blacksburg, Virginia (N.C.)
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Martin A. Hayes
Cardiovascular and Metabolic Diseases(X.-Q.L., M.A.H., L.W.) and Respiratory, Inflammation, and Autoimmune Disease (G.G., K.B.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; and Department of Chemistry, Virginia Tech, Blacksburg, Virginia (N.C.)
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Gunnar Grönberg
Cardiovascular and Metabolic Diseases(X.-Q.L., M.A.H., L.W.) and Respiratory, Inflammation, and Autoimmune Disease (G.G., K.B.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; and Department of Chemistry, Virginia Tech, Blacksburg, Virginia (N.C.)
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Kristina Berggren
Cardiovascular and Metabolic Diseases(X.-Q.L., M.A.H., L.W.) and Respiratory, Inflammation, and Autoimmune Disease (G.G., K.B.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; and Department of Chemistry, Virginia Tech, Blacksburg, Virginia (N.C.)
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Neal Castagnoli Jr.
Cardiovascular and Metabolic Diseases(X.-Q.L., M.A.H., L.W.) and Respiratory, Inflammation, and Autoimmune Disease (G.G., K.B.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; and Department of Chemistry, Virginia Tech, Blacksburg, Virginia (N.C.)
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Lars Weidolf
Cardiovascular and Metabolic Diseases(X.-Q.L., M.A.H., L.W.) and Respiratory, Inflammation, and Autoimmune Disease (G.G., K.B.), Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden; and Department of Chemistry, Virginia Tech, Blacksburg, Virginia (N.C.)
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  • Fig. 1.
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    Fig. 1.

    High resolution product ion mass spectra acquired by collision-induced dissociation of the respective MH+ ions of AZD1979 (top) and M1 (bottom). Insets are the tentative fragmentation pattern and the full-scan MS spectra.

  • Fig. 2.
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    Fig. 2.

    Proposed potential structure (1 or 2) of hydrated metabolite M1 of AZD1979.

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    Fig. 3.

    NAD(P)H dependence of the formation of M1. AZD1979 (10 µM) was incubated with various human liver subcellular fractions (1 mg/ml protein), with and without the presence of a 1-mM mixture of NADPH-NADH over 60 minutes. Results are averages of duplicate measurements. Mean of M1 formation in HLM incubations with and without NAD(P)H was set as 100% abundance.

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    Fig. 4.

    HRMS spectra of M1 in human liver S9 fractions incubated with AZD1979 (10 µM) for 120 minutes, with (A) and without (B) H218O (77.6% 18O) enrichment. The inset shows the time course of the accumulated incorporation of 18O in M1 in human liver S9 fractions or HLMs.

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    Fig. 5.

    Effect of pH on M1 formation in the incubation of AZD1979 (50 µM) with HLMs or S9 fractions over 60 minutes. Results are averages of duplicate measurements.

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    Fig. 6.

    Kinetics of the formation of M1 in HLMs. A range of AZD1979 concentrations were incubated in HLMs at 37°C for 30 minutes. The velocity (picomoles per minute per milligram protein) versus concentration curve was fitted to a single-site Michaelis-Menten equation.

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    Fig. 7.

    Effect of progabide on M1 formation after incubation of AZD1979 (10 µM) in human liver S9 fractions, cytosol, and microsomes. Results are averages of duplicate measurements.

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    Fig. 8.

    Inhibition effect of progabide on M1 formation after 30-minute incubation of AZD1979 (200 µM) in HLMs. Activity is expressed as a percentage of control activity measured in the absence of inhibitor. IC50 values were determined by nonlinear regression.

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    Fig. 9.

    Proposed pathways for the mEH-catalyzed hydration of the oxetanyl moiety of AZD1979 and related incorporation of oxygen from water into mEH and product diol. The single-turnover and the subsequent reactions are shown to illustrate the origin of the additional oxygen incorporated in the diol product. The oxygen atom originating from mEH is shown in blue and that from water in the medium is in red. In the hydroxylalkyl-enzyme intermediate, the new covalent bond formed is in yellow and the bond cleaved to release the final diol product is in green. H+ in the cycle represents proton transfer steps that are not shown.

Additional Files

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  • Data Supplement

    Files in this Data Supplement:

    • Supplemental Data -

      NMR characterization of synthesized compound AZ13478123 Methods

      Supplemental Figure 1 - 600 MHz 1H spectra of AZ13478123 (top) and AZD1979 ( bottom) in CD3OD at 25 deg

      Supplemental Figure 2 - 150 MHz 13C spectra of AZ13478123 (top) and AZD1979 (bottom) in CD3OD at 25 deg

      Supplemental Figure 3 - 1H -13C HMBC long-range correlation from CH2(5,7) to CH2(8) of AZ13478123

      Supplemental Table 1 - NMR assignments for 3-(4-{3,3-bis(hydroxymethyl)azetidin-1- ylmethyl}phenoxy)azetidin-1-yl5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-ylmethanone (AZ13478123) and AZD1979

      Supplemental Figure 4 - Comparison of extracted ion chromatograms of AZD1979 and M1 (mass tolerance 10 ppm) in samples without (A and C) and with (B and D) the spiking of the synthesized diol, AZ13478123

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Drug Metabolism and Disposition: 44 (8)
Drug Metabolism and Disposition
Vol. 44, Issue 8
1 Aug 2016
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Research ArticleSpecial Section on Emerging Novel Enzyme Pathways in Drug Metabolism

Microsomal Epoxide Hydrolase–Catalyzed Hydration of an Oxetane

Xue-Qing Li, Martin A. Hayes, Gunnar Grönberg, Kristina Berggren, Neal Castagnoli and Lars Weidolf
Drug Metabolism and Disposition August 1, 2016, 44 (8) 1341-1348; DOI: https://doi.org/10.1124/dmd.116.071142

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Research ArticleSpecial Section on Emerging Novel Enzyme Pathways in Drug Metabolism

Microsomal Epoxide Hydrolase–Catalyzed Hydration of an Oxetane

Xue-Qing Li, Martin A. Hayes, Gunnar Grönberg, Kristina Berggren, Neal Castagnoli and Lars Weidolf
Drug Metabolism and Disposition August 1, 2016, 44 (8) 1341-1348; DOI: https://doi.org/10.1124/dmd.116.071142
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